Methods for preventing, modulating and/or reducing cardiovascular disease

ABSTRACT

Methods and materials for preventing and modulating atherosclerosis. In particular, small peptides that are capable of interacting with CD40, thereby interfering with the ability of CD40 to interact with CD154, which impacts inflammation and atherosclerosis. The use of such peptides in reducing atherosclerosis, and in particular, the autoimmune inflammatory response that may he a driving factor thereof. The use of such short peptides to lower LDL cholesterol. Methods and materials for detecting T-cells that express CD40 (Th40 cells). Methods and materials for confirming or ruling out cardiovascular disease, atherosclerosis, and/or coronary artery disease.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file entitled “US_16_528269_Sequence_Listing.txt” having a size inbytes of 12 kb and created Nov. 22, 2019. The information contained inthis electronic file is hereby incorporated by reference in its entiretypursuant to 37 CFR 1.52(e)(5).

This invention was made with government support under grant number42DK115296-02 and R42AI131784-02 awarded by the National Institutes ofHealth. The government may have certain rights in the invention.

The present disclosure relates to methods or uses for prevention,modulation, and reduction of cardiovascular disease and/oratherosclerosis in a subject in need of a therapeutically effectiveamount of a CD40 peptide that inhibits the interaction of CD40 andCD154, and the use of such compounds in modulating T-cell activity andin treating disease. Furthermore, this disclosure relates to methods andmaterials for confirming or ruling out cardiovascular disease,atherosclerosis, and/or coronary artery disease.

BACKGROUND

According to the World Health Organization (WHO), an estimated 17.7million people died from cardiovascular diseases (CVDs) in 2015, whichrepresented 31% of all global deaths. CVDs, may also be known as heartand blood vessel disease, and includes numerous problems, many of whichare related to a process called atherosclerosis. Moreover, according tothe Healthcare Cost and Utilization Project (HCUP) which is sponsored bythe Agency for Healthcare Research and Quality (AHRQ), in 2011, coronaryatherosclerosis alone accounted for more than $10.4 billion in hospitalcosts. Accordingly, in 2011, coronary atherosclerosis alone was one ofthe ten most expensive conditions for inpatient hospitalizations in theUnited States.

Atherosclerosis is defined by arterial plaque formation that may lead toheart attack and stroke. Arterial plaque formation is caused by thedeposition of cells, substances, waste products, and cellular debrisincluding, but not limited to: cholesterol, dead cells, dendritic cells,foam cells, macrophages, mast cells, monocytes, smooth muscle cells,T-cells, collagen, calcium, and fibrin. Inflammatory changes within thearterial wall and plaque may play a crucial and causative role inatherosclerotic disease development. Consequently, the concept ofatherosclerosis as an autoimmune and inflammatory disease has beeninvestigated; however, a therapeutic control has not been established.The importance of controlling inflammation is highlighted by currentclinical trials targeting other aspects of autoimmune, inflammation, andcardiovascular disease and death.

For example, CIRT (Cardiovascular Inflammation Reduction Trial) isattempting to use methotrexate to target interleukin-6 (IL-6) to testwhether methotrexate will reduce rates of myocardial infarction, stroke,and cardiovascular death among patients with coronary artery diseasepatients with type 2 diabetes. Another example includes, CANTOS(Canakinumab Anti-inflammatory Thrombosis Outcomes Study) which isstudying whether canakinumab can block the pro-inflammatory cytokineinterleukin-1β (IL-1β) to reduce rates of recurrent myocardialinfarction, stroke, and cardiovascular death amount heart attackpatients who remain at a high risk because of elevated levels of theinflammatory biomarker high sensitivity C-reactive protein (hsCRP).These studies acknowledge that inflammation plays a critical role inatherothrombosis; however, these studies also recognize that is unknownwhether inhibition of inflammation per se will lower vascular eventrates.

Mammalian and human atherosclerotic lesions are characterized as achronic inflammatory-fibroproliferative disease of the blood vessel wallcontaining monocytes, macrophages, endothelial cells, smooth musclecells, platelets, and T-cells. Each of these cell types can expresseither or both of the CD40/CD154 costimulatory pair. This dyad isresponsible for enhancing the immune response and may contribute to manychronic inflammatory diseases including rheumatoid arthritis, multiplesclerosis, and type 1 diabetes (T1D). However, no viable therapy existsfor this highly atherogenic dyad.

Inflammation may occur when inflammatory cells, such as neutrophils,eosinophils, basophils, mast cells, macrophages, platelets, andendothelial cells, respond to inflammatory events or harmful stimuli,such as, invading microorganisms, damages cells, or other irritants. Thebody's inflammatory response is beneficial because for example, in thecase of invading microorganisms, the inflammatory response is animportant step in localizing the infecting agent for removal by theimmune system. However, in autoimmunity there is no infection, yetsevere inflammation is present or persistent. The inflammation in thiscase, referred to as aseptic chronic inflammation (ACI), is detrimentalsince it destroys normal tissues. The results of this asepticinflammation are life-altering and in some cases life-threatening.Moreover, as with acute inflammation, this process is mediated by immunecells, including T-cells.

A major concern for modern medicine is how to control ACI such as thatwhich occurs during autoimmune diseases, as well as how to control acuteinflammation resulting from trauma. Inflammation, both chronic andacute, leads to tissue degeneration and eventual loss of function ofmajor organs. ACI is not limited to a single disease, but isinstrumental in numerous autoimmune diseases, including, but not limitedto: type 1 diabetes (T1D), multiple sclerosis, systemic lupuserythematosus, rheumatoid arthritis, Crohn's disease, inflammatory boweldisease, chronic obstructive pulmonary disease including types ofautoimmune asthma, atherosclerosis, vasculitis, hypertension,thyroiditis including Hashimoto's and Graves diseases, primary biliarycirrhosis, Paget's disease, Addison's disease, acute respiratorydistress syndrome, acute lung injury, and ACI associated with organtransplantation.

Autoimmune disorders are classified into two types: organ-specific(directed mainly at one organ) and non-organ-specific (widely spreadthroughout the body). Examples of organ-specific autoimmune disordersare insulin-dependent Type 1 diabetes (T1D) which affects the pancreas;Hashimoto's thyroiditis and Graves' disease, which affect the thyroidgland; pernicious anemia, which affects the blood; Addison's disease,which affects the adrenal glands; chronic active hepatitis, whichaffects the liver; myasthenia gravis which affects the muscle; andmultiple sclerosis, which affects tissue of the nervous system. Anexample of a non-organ-specific autoimmune disorders is rheumatoidarthritis. Autoimmune diseases are often chronic, debilitating, andlife-threatening. The National Institutes of Health (NIH) estimates thatup to 23.5 million Americans suffer from autoimmune disease and that theprevalence is rising. It has been estimated that autoimmune diseases areamong the ten leading causes of death among women in all age groups upto 65 years.

Acute inflammation, as observed during trauma or sepsis, is also immunecell mediated. While a comprehensive, complete, and exhaustive list ofthe molecular mediators in this process have not yet been identified, aprominent role for T-cells, lymphocytes, neutrophils, macrophages,monocytes, neutrophils, eosinophils, basophils, mast cells, and otherinflammatory cells is strongly implicated. Therefore, a process tomodulate these cell types may control the inflammatory response.

A unique T cell subset has been shown to be instrumental in thedevelopment of autoimmune disease. These cells are phenotypicallycharacterized as CD4loCD40+(Waid, D. M., G. M. Vaitaitis, and J. Wagner,D. H. 2004. European Journal of Immunology 34:1488; Vaitaitis, G. M., M.Poulin, R. J. Sanderson, K. J. Haskins, and D. H. Wagner Jr. 2003.Cutting Edge, J. Immunol. 170:3455; Wagner, D. H., Jr., G. Vaitaitis, R.Sanderson, M. Poulin, C. Dobbs, and K. Haskins. 2002. Proc Natl Acad SciUSA 99:3782; Wagner, D. H., Jr., E. Newell, R. Sanderson, J. H. Freed,and M. K. Newell. 1999. International Journal of Molecular Medicine4:231), and are referred to as Th40 cells. (Waid, D. M., et al. (2004)Eur. J. of Immunol. 34:1488; Vaitaitis, G. M., et al. 2003. CuttingEdge, J. Immunol. 170:3455; Wagner, D. H., Jr., et al. (2002) Proc.Nat'l. Acad. Sci. USA 99:3782; Wagner, D. H., Jr., et al. (1999) Int'lJ. of Mol. Med. 4:231). Th40 cells may be any CD4+ T cell thatco-expresses CD40 and CD4hi or CD4lo. CD40 expression typically isassociated with antigen presenting cells and the majority of prior artdescribes CD40 as being expressed on B cells, macrophages, monocytes,and other cells; however, CD40 proteins are also expressed on T cells(Waid, D. M., et al., G. M. Vaitaitis, and J. Wagner, D. H. 2004.European Journal of Immunology 34/1488; Vaitaitis, G. M., M. Poulin, R.J. Sanderson, K. J. Haskins, and D. H. Wagner Jr. 2003. Cutting Edge, J.Immunol. 170-3455; Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M.Poulin, C. Dobbs, and K. Haskins. 2002. Proc Natl Acad Sci USA 99:3782;Wagner, D. H., Jr., E. Newell, R. Sanderson, J. H. Freed, and M. K.Newell. 1999. International Journal of Molecular Medicine 4:231;Bourgeois, C., B. Rocha, and C. Tanchot. 2002. Science 297:2060;Fanslow, W. C., K. N. Clifford, M. Seaman, M. R. Alderson, M. K.Spriggs, R. J. Armitage, and F. Ramsdell. 1994. Journal of Immunology152:4262; Ramsdell, F., M. S. Seaman, K. N. Clifford, and W. C. Fanslow.1994. Journal of Immunology 152:2190; Grabstein, K. H., C. R.Maliszewski, K. Shanebeck, T. A. Sato, M. K. Spriggs, W. C. Fanslow, andR. J. Armitage. 1993. Journal of Immunology 150:3141; Armitage, R. J.,C. R. Maliszewski, M. R. Alderson, K. H. Grabstein, M. K. Spriggs, andW. C. Fanslow. 1993. Seminars in Immunology 5:401; Cooper, C. J., G. L.Turk, M. Sun, A. G. Farr, and P. J. Fink. 2004. J Immunol 173:6532).While Th40 cells comprise a proportion of the peripheral CD4+compartment in naïve, non-autoimmune mice (Waid, D. M., G. M. Vaitaitis,and J. Wagner, D. H. 2004. European Journal of Immunology 34:1488;Wagner, D. H., Jr., E. Newell, R. Sanderson, J. H. Freed, and M. K.Newell. 1999. International Journal of Molecular Medicine 4:231), and inhumans (Waid. D. M, R. J. Wagner, A. Putnam, G. M. Vaitaitis, N. D.Pennock, D. C. Calverley, P. Gottlieb, and D. H. Wagner, Jr. 2007. ClinImmunol 124:138), this proportion is drastically expanded to as much as50% of the CD4+ compartment in autoimmune prone mice (Waid, D. M., G. M.Vaitaitis, and J. Wagner. 2004. European Journal of Immunology 34:1488;Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, andK. Haskins. 2002. Proc Natl Acad Sci USA 99:3782; Wagner, D. H., Jr., E.Newell, R. Sanderson, J. H. Freed, and M. K. Newell. 1999. InternationalJournal of Molecular Medicine 4:231) and humans (Waid, D. M., et al.(2004) Eur. J. of Immunol. 34:1488; Waid. D. M. et al. (2007) Clin.Immunol. 124:138). (Waid. D. M, R. J. Wagner, A. Putnam, G. M.Vaitaitis, N. D. Pennock, D. C. Calverley, P. Gottlieb, and D. H.Wagner, Jr. 2007. Clin Immunol 124:138). These T cells do not expressearly activation markers and occur in the naïve phenotype ofnon-challenged mice.

In NOD (non-obese diabetic) mice, Th40 cells occur at exaggerated levelsin spleen, lymph nodes and the pancreas, even prior to diabetes onset(Waid, D. M., et al. (2004) Eur. J. of Immunol. 34:1488 Waid, D. M., G.M. Vaitaitis, and J. Wagner, D. H. 2004. European Journal of Immunology34:1488).; Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M. Poulin, C.Dobbs, and K. Haskins. 2002. Proc Natl Acad Sci USA 99:3782). Anelevated number and percentage of these T cells are seen in peripheralblood of type 1 diabetic (T1D) patients when compared to non-autoimmunecontrols and type 2 diabetic patients (Waid. D. M., et al. (R. J.Wagner, A. Putnam, G. M. Vaitaitis, N. D. Pennock, D. C. Calverley, P.Gottlieb, and D. H. Wagner, Jr. 2007. Clin Immunol 124:138).

The observed increase in Th40 cells could mean that those T cells areantigen responsive or that CD40 expression is activation induced.Furthermore, several diabetogenic T cell clones are CD40+ (Wagner, D.H., Jr., et al. (G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, and K.Haskins. 2002). Proc. Nat'l. Acad. Sci. USA 99:3782). Purified primaryTh40 cells from NOD mice and from pre-diabetic NOD (12-weeks of age)mice successfully transfer type 1 diabetes to NOD/scid (Non-ObeseDiabetic/Severe Combined Immunodeficiency) recipient mice, directlydemonstrating pathogenicity of the Th40 T cell subset (Waid, D. M., etal. (2004) Eur. J. of Immunol. 34:1488 Waid, D. M., G. M. Vaitaitis, andJ. Wagner, D. H. 2004. European Journal of Immunology 34:1488). Further,Wagner, D. H., Jr., G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, andK. Haskins. 2002. Proc Natl Acad Sci USA 99:3782). It has been shownthat Th40 cells infiltrate islet beta cells destroying insulinproduction thus suggesting islet antigen specificity (Waid, D. M., etal. (2004) Eur. J. of Immunol. 34:1488 Waid, D. M., G. M. Vaitaitis, andJ. Wagner, D. H. 2004. European Journal of Immunology 34:1488). Wagner,D. H., Jr., G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, and K.Haskins. 2002. Proc Natl Acad Sci USA 99:3782). It has also been shownthat Th40 cells are required for diabetes transfer. Peripheral (spleenand regional lymph node) T cells that were CD40 depleted, then CD25,Treg, depleted were not capable of transferring diabetes to Scid (SevereCombined Immunodefienciency) recipients. Even though Treg cells wereremoved, if the auto-aggressive CD40+ T cells subset is absent, diseasetransfer does not occur.

While Th40 cells are important in the development of autoimmunity,another important factor is expression of the CD40-Ligand, CD154. CD154is temporally induced on activated T-cells in response to CD3/TCRstimulation (Lederman, S. et al. (M. Yellin, A. Krichevsky, J. Belko, J.Lee, and L. Chess. 1992) Journal of Experimental Medicine 175:1091).CD154 expression has also been demonstrated on platelets, monocytes,basophils, eosinophils, dendritic cells, fibroblasts, smooth muscle, andendothelial cells (Russo, S. et al. (B. Bussolati, I. Deambrosis, F.Mariano, and G. Camussi, 2003). J. Immunol. 171:5489; Stumpf, C., etal., (C. Lehner, S. Eskafi, D. Raaz, A. Yilmaz, S. Ropers, A.Schmeisser, J. Ludwig, W. G. Daniel, and C. D. Garlichs. 2003). Eur. J.Heart Fail. 5:629; Schonbeck, U., et al., and P. Libby. (2001) Cell Mol.Life Sci. 58:4). CD154 is a member of the tumor necrosis factor (TNF)super-family and a soluble form of CD154 (sCD154) has been described(Russo, S. et al. B. Bussolati, I. Deambrosis, F. Mariano, and G.Camussi. 2003). J. Immunol. 171:5489; Stumpf, C., et al., (C. Lehner, S.Eskafi, D. Raaz, A. Yilmaz, S. Ropers, A. Schmeisser, J. Ludwig, W. G.Daniel, and C. D. Garlichs. 2003). Eur. J. Heart Fail 5:629; Toubi, E.,et al. (2004) and Y. Shoenfeld. 2004 Autoimmunity 37:457). Therefore,sCD154 may act like a cytokine (Stumpf, C., C. Lehner, S. Eskafi, D.Raaz, A. Yilmaz, S. Ropers, A. Schmeisser, J. Ludwig, W. G. Daniel, andC. D. Garlichs et al (2003) Eur. J. Heart Fail. 5:629). Even thoughCD154 has not been genetically linked in T1D studies, sCD154 issignificantly elevated in T1D and may play a role in the disease process(Varo, N. et al. (D. Vicent, P. Libby, R. Nuzzo, A. L. Calle-Pascual, M.R. Bernal, A. Fernandez-Cruz, A. Veves, P. Jarolim, J. J. Varo, A.Goldfine, E. Horton, and U. Schonbeck. 2003) Circulation 107:2664).Cipollone, F., F. Chiarelli, G. Davi, C. Ferri, G. Desideri, M. Fazia,A. lezzi, F. Santilli, B. Pini, C. Cuccurullo, S. Tumini, A. Del Ponte,A. Santucci, F. Cuccurullo, and A. Mezzetti. 2005. Diabetologia 48:1216;Devaraj, S., N. Graser, S. Griffen, J. Wang-Polagruto, E. Miguelino, andI. halal. 2006. Diabetes 55:774). The importance of CD40-CD154interaction in autoimmunity has been established (Wagner, D. H., Jr., etal. (G. Vaitaitis, R. Sanderson, M. Poulin, C. Dobbs, and K. Haskins.2002) Proc. Nat'l. Acad. Sci. USA 99:3782; Kobata, T., et al. M. Azuma,H. Yagita, and K. Okumura. (2000) Rev. Immunogenet. 2:74; Homann, D., etal., A. Jahreis, T. Wolfe, A. Hughes, B. Coon, M. J. van Stipdonk, K. R.Prilliman, S. P. Schoenberger, and M. G. von Herrath. (2002) Immunity16:403; Goodnow, C. C. et al., (2001) Lancet 357:2115; Balasa, B., etal., (1997) T. Krahl, G. Patstone, J. Lee, R. Tisch, H. O. McDevitt, andN. Sarvetnick. 1997. J. of Immunol. 159:4620). Blocking CD40-CD154interaction may prevent collagen induced arthritis, (Durie, F. H., R. A.Fava, T. M. Foy, A. Aruffo, J. A. Ledbetter, and R. J. Noelle. 1993.Science 281:1328) experimental autoimmune encephalitis (Howard, L. M.,and S. D. Miller. 2004. Autoimmunity 37:411), prostatitis (Grossman, M.E., E. Davila, and E. Celis. 2001. J Immunother 24:237), and type-1diabetes in the NOD mouse model (Durie, F. H. et al. (1993) Science281:1328; Balasa, B. et al. (T. Krahl, G. Patstone, J. Lee, R. Tisch, H.O. McDevitt, and N. Sarvetnick. 1997) Journal of Immunology 159:4620;Howard, L. M. et al. (2004) Autoimmunity 37:411; Grossman, M. E. et al.(2001) J. Immunother. 24:237). In the diabetes model, it was essentialto administer a CD154 blocking antibody to NOD mice at 3-weeks of agebecause at 9-weeks, blocking antibodies had no effect on diabetesprevention (Balasa, B. et al. (T. Krahl, G. Patstone, J. Lee, R. Tisch,H. O. McDevitt, and N. Sarvetnick. 1997) Journal of Immunology159:4620).

Previous work has also demonstrated that the Th40 cell subset inducesRAG1 and RAG2 (Recombination-Activating Genes) transcription,translation and nuclear translocation (Vaitaitis, G. M., M. Poulin, R.J. Sanderson, K. J. Haskins, and D. H. Wagner Jr. 2003. Cutting Edge, J.Immunol. 170:3455) when CD40 is engaged (Vaitaitis, G. M. et al. (2003)Cutting Edge, J. Immunol. 170:3455). CD3 engagement does not induce RAG1or RAG2 in T-cells (Vaitaitis, G. M., et al. (M. Poulin, R. J.Sanderson, K. J. Haskins, and D. H. Wagner Jr. 2003) Cutting Edge, J.Immunol. 170:3455). Subsequent to RAG1/RAG2 induction, CD40-mediatedT-cell receptor (TCR) revision occurs in peripheral T cells (Vaitaitis,G. M. et al., M. Poulin, R. J. Sanderson, K. J. Haskins, and D. H.Wagner Jr. 2003. Cutting Edge, J. Immunol. 170:3455). CD40 induction ofTCR revision is RAG dependent. T cells isolated from a TCR-Tg mouseundergo TCR revision when CD40 engaged, but T-cells from theTCR-Tg.RAG−/− mouse do not TCR revise when CD40 engaged (Wagner, D. H.,Jr. et al. , E. Newell, R. Sanderson, J. H. Freed, and M. K. Newell.(1999) Int'l J. of Mole. Med. 4:231).

CD40 is a 50-kDa integral membrane protein of the tumor necrosis factorreceptor (TNF-R) family. It is constitutively expressed as a homotrimer(Foy T M, et al. (1996) Annu. Rev. of Immunol., 14:591-617). In general,stimulation of all CD40-expressing cell types induces operations whichcontribute to inflammation, such as enhancement of costimulatory andadhesion molecules, and up-regulation of proteolytic enzymes (Mach, F.et al. (1998) Atherosclerosis. 137 Suppl:S89-95). Moreover,CD40-expressing cell types may induce operations which contribute toinflammation, such as enhancement of costimulatory and adhesionmolecules, activation of chemokine and cytokine synthesis andup-regulation of proteolytic enzymes. (Mach, F., et al., (1998)Atherosclerosis. 137 Suppl: S89-95).

CD40's ligand—CD154—is a 39-kDa protein that belongs to the tumornecrosis factor (TNF) family. CD40 forms a trimer that binds CD154 atthe interface of the three monomers. CD154 is expressed commonly oncells beyond the surface-expressed CD154, as CD154 may also exist in asoluble biologically active form (sCD154) that is shed from the cellsurface after activation. The main source of sCD154 is platelets. (Foy TM, et al. (1996) Annu. Rev. of Immunol., 14:591-617).

Genetically manipulated mouse models are utilized for research anddevelopment concerning atherosclerosis because wild type mice aregenerally highly resistant to development and progression ofatherosclerosis. Prior studies have attempted to block the CD40/CD154interaction by using mono-clonal antibodies and this approach has provenefficacious in several mouse model studies utilizing the Apoe−/− or LDLrdeficient atherosclerotic models. Additionally, these same mouse modelsbuilt with a deletion of CD154 saw significant reductions in overallplaque formation and may have also contributed to production of a morestable plaque phenotype. Clinically, stable plaques are identifiable anddenoted by increased collagen and smooth muscle content, a thick fibrouscap, and an observable decrease in T cell, macrophage, and lipidaccumulation.

Multiple treatment options have been put forward to address and controlboth chronic and acute inflammation. Many approaches use non-steroidalanti-inflammatory drugs (NSAIDS) that attack the production ofleukotrienes and prostaglandins, cellular products that cause localizedinflammation. Other approaches use more powerful immunosuppressant drugssuch as cyclophosphamide, methotrexate and azathioprine that suppressthe immune response and stop the progression of the disease. Still othertreatments involve the use of monoclonal antibodies designed to alterthe immune responses to self-tissues, as occurs during autoimmunediseases. However, all of these treatments often have severe, long-termside effects.

Current immune-modulatory therapies may rely upon monoclonal antibodytreatments that may give rise to complications. For example, antibodiesadministered to a subject may cross-react with unintended targets andcause severe nephritic complications and those that specifically actagainst CD154 may cause embolic complications. Further, the CD40-CD154interaction may play an important role in antibody generation which mayindicate that administration of a monoclonal antibody could induceauto-antibody generation and further complications, which may inhibitthe restoration of normal immune function (see generally Banchereau, J.et al. (1994) Annu. Rev. of Immunol. 12:881-920).

Other studies have demonstrated that blocking the CD154 interaction byusing mono-clonal antibodies, or limiting the CD40 receptor bymono-clonal antibodies may abrogate atherosclerosis, and may confer amore favorable plaque phenotype characterized by lower inflammation andhigher fibrosis. These studies additionally demonstrated that neointimalformation and restenosis may be limited by blocking the CD154interaction. Studies concerning lupus nephritis may have demonstratedthat blocking CD40 mediated signals can reduce anti-double-stranded DNA(anti-dsDNA) antibodies. Moreover, these studies may demonstrate thatthe reduction of anti-DNA was associated with increased serum complementlevels and reduced glomerular inflammation, which may be viewedpositively from a clinical perspective. However, the use of mono-clonalantibodies to target the CD40/CD154 dyad was abandoned due tothromboembolic events which may have been related to the functioning ofCD154 in thrombus stabilization. It is postulated that CD154 stabilizethrombi by interaction with the integrin α_(IIb)β₃, and by inhibitingCD154, thrombi may be less stable, and as a consequence shed embolicausing thrombotic events.

Currently, Coronary Artery Calcium (CAC) scores are used in detectingcoronary artery disease; however, current method for developing calciumscores using radiation are expensive, invasive, and time consuming.

Thus, there exists a need in the art for safer and more effectivemethods for treatment and prevention of cardiovascular diseases (CVDs)implicated by aseptic chronic inflammation (ACI). The presentdevelopments may address this need by describing methods for treatmentof atherosclerosis by administration of a therapeutically effectiveamount of CD40 peptide. Further, the present developments may providethe added benefit of preventing auto-antibody generation, and thus allowthe resumption of normal immune function. Also, there exists a need toreduce the use of CAC scores based on methods that use radiation andinvasive procedures.

This statement of background is for information purposes only and is notintended to be a complete or exhaustive explication of all potentiallyrelevant background.

SUMMARY

The present developments may provide a novel methods for preventing,modulating, and/or reducing atherosclerosis that arises in a corporealbody. Atherosclerosis may arise as a result of chronic inflammatoryresponse of white blood cells in the walls of arteries. It is postulatedthat the chronic inflammatory response may and the subsequent buildupsof plaque in arteries may be caused by elevated levels of cholesteroland triglycerides in the blood, high blood pressure, and cigarettesmoking.

The present developments are based on the knowledge that interaction ofCD40-ligand (CD154 protein) with CD40 protein expressed on T-cells (Th40cells), may be important in the development of atherosclerosis andautoimmune disease. The present developments may be based on theelucidation of the critical residues in CD40 and CD154 that may beimportant for this interaction. The present developments relate toaffecting the interaction between a CD40 protein and a CD154 proteinthrough the use of small peptides that interact with the CD40 protein ata site where the CD154 protein would normally bind. The presentdevelopments also relate to using such peptides to reduce the level ofTh40 cells, thereby reducing the severity of disease.

One embodiment of the present developments is a method for preventingatherosclerosis comprising contacting the CD40 protein with a peptidethat interacts with the CD40 protein. Preferred peptides are those thatare less than 25 amino acids in length, and that bind to a CD40 protein,thereby inhibiting its interaction with a CD154 protein.

One embodiment of the present developments is a method for preventing,modulating, and/or reducing atherosclerosis, the method comprisinginhibiting interaction between a CD40 protein and a CD154 protein with apeptide that interacts with the CD40 protein. Preferred peptidesinteract with the CD40 protein at the CD154-binding site. Preferablysuch peptides are less than 25 amino acids in length. Even morepreferred peptides are those amino acid sequences selected from SEQ IDNOs 3-10.

One embodiment of the present developments is a method to modulateand/or reduce atherosclerosis in an animal, the method comprisingadministering to the animal, a peptide that interacts with a CD40protein in such a manner as to modulate INFγ. Preferred peptides arethose that interact with the CD40 protein at the CD154binding site,thereby modulating INFγ. Preferred peptides modulate inflammation byreducing the level of Th40 cells to no more than 25% of the total T-cellpopulation. Such methods can be used to prevent and/or reduceatherosclerosis and symptoms that may accompany cardiovascular diseases,more generally.

One embodiment of the present developments is a method to identify apatient at risk for developing cardiovascular disease and/oratherosclerosis, the method comprising obtaining a sample containingT-cells from a patient to be tested, contacting the sample with apeptide that binds the CD40 protein, detecting the CD-40 bound peptide,and determining the level of Th40 cells from the amount of CD40 bound,wherein a level of Th40 cells greater than 25% of the total T-cellpopulation indicates the patient is at risk for developingcardiovascular disease and/or developing atherosclerosis.

Yet, another embodiment of the present developments is a method toprevent, modulate, or reduce calcium buildup, or calcification of vesselwalls, the method comprising administering to the subject in needthereof, a therapeutically effective amount of a peptide whichspecifically binds to a CD40 presenting cells at the CD154 binding site.

Another embodiment of the present developments, is a method toadminister a CD40 peptide to prevent, modulate, and/or reduceatherosclerosis, comprising selecting a peptide that interacts with aCD40 protein and CD154 binding site, selecting a delivery methodselected from the group comprising intramuscular (IM) delivery,intravenous (IV) delivery, subcutaneous (SC) delivery, oral delivery,gavage delivery, emolument/skin delivery, or transdermal patch.

Another embodiment of the present developments, is a method toadminister a CD40 peptide to prevent, modulate, and/or reduceatherosclerosis in an animal, comprising selecting a peptide thatinteracts with a CD40 protein and CD154 binding site, using an extendeddelivery method selected from the group comprising an implantabledevice, a hydrophilic polymer formulation, a permeable polymericmembrane, injectable gel implants, solvent extraction system, phaseinversion system, thermosensitive gels, pH dependent in situ gels,microparticles, microspheres, nanoparticles, nanospheres, bio-degradableimplants, or photoactivated depot.

Another embodiment of the present developments, is a method to lower LDLcholesterol in a subject, the method comprising administering to thesubject in need thereof, a therapeutically effective amount of a peptidewhich specifically binds to a CD40 presenting cells at the CD154 bindingsite.

Another embodiment of the present developments is a method to measureTh40 cell levels and compare to known coronary artery calcium scores fora population subset to provide an accurate, non-invasive, radiationfree, and alternative method of detecting when a subject with T1D may beat risk of coronary artery disease.

Another embodiment of the present developments is a method to confirm orrule out diagnosis of cardiovascular disease, atherosclerosis, and/orcoronary artery disease in a patient comprising: determining thepercentage of Th40 cells in a sample isolated from a subject presentingwith symptoms suggestive of cardiovascular disease, atherosclerosis,and/or coronary artery disease, comparing the percentage of Th40 cellsto a control sample or a standard value, and diagnosing cardiovasculardisease, atherosclerosis, and/or coronary artery disease in the subjecthaving an increase in the percentage of Th40 cells in the sample fromthe subject relative to the control sample or standard value isindicative of cardiovascular disease, atherosclerosis, and/or coronaryartery disease in the subject.

Another embodiment of the present developments is a method to confirm orrule out diagnosis of cardiovascular disease, atherosclerosis, and/orcoronary artery disease in a patient comprising: determining thepercentage of Th40 cells in a sample isolated from a subject,determining the percentage of interferon gamma (IFNγ) in a sampleisolated from a subject, comparing the percentage of Th40 cells to acontrol sample or a standard value, comparing the percentage ofinterferon gamma (IFNγ) to a control sample or standard value, anddiagnosing cardiovascular disease and/or coronary artery disease in thesubject having an increase in the percentage of Th40 cells andinterferon gamma (IFNγ) in the sample from the subject relative to thecontrol sample or standard value; or, ruling out cardiovascular disease,atherosclerosis, and/or coronary artery disease in the subject having noincrease or a decrease in the percentage of Th40 cells and interferongamma (IFNγ) in the sample from the subject relative to the controlsample or standard value.

Another embodiment of the present developments is a method foridentifying and treating a patient for cardiovascular disease, themethod comprising: detecting the percentage of Th40 cells in T-cells andthe level of interferon gamma (IFNγ) of the Th40 cells in a blood samplefrom the patient; and treating the patient with elevated percentage ofTh40 cells and interferon gamma (IFNγ) relative to a percentage of Th40cells and interferon gamma (IFNγ) in a blood sample from a normalcontrol sample with a drug in an amount to decrease the percentage ofthe Th40 cells in the patient's blood.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a chart of the effect of various peptides of CD154 on thedevelopment of diabetes in NOD mice. The 6-mer (SEQ ID NO: 4), 8-mer(SEQ ID NO: 5), 10-mer (SEQ NO: 24), 13-mer (SEQ ID NO:25), 15-mer (SEQID NO: 7), and 24-mer (SEQ ID NO:26) were tested.

FIG. 2A is a chart of the effect of a 15-mer peptide from CD154 on theCD4/CD8 ratio in NOD mice.

FIG. 2B is a chart of the effect of 15-mer peptide in treated versuscontrol pancreata excised, examined, and scored.

FIG. 3 is a graph of reversal of diabetes in NOD mice using a 15-merpeptide from CD154.

FIG. 4 is a dot-plot of the detection of Th40 cells using a 15-merpeptide from CD154.

FIG. 5 is a dot-plot of a screening of B cells using a 15-mer peptidefrom CD154.

FIG. 6 is a chart demonstrating a comparison of Th40 cell levels indiabetic and non-diabetic mice.

FIG. 7 is a chart demonstrating the effect of treatment with the 15-merpeptide on insulin granulation of the pancreas.

FIG. 8 is a graph that shows the effect of mutations in the 15-merpeptide on the ability of the 15-mer peptide to inhibit development ofdiabetes in NOD mice.

FIG. 9 is a chart showing the number of cells (×10⁶) of CD3+CD4+CD40+ indifferent mice models.

FIG. 10 is a chart showing the percentage of TH40 cells in theperipheral blood in human subjects in control and diabetic subjects.

FIG. 11 is a dot-plot comparing CD4+ and CD40 cell data obtained throughflow cytometry.

FIG. 12 is a chart showing the percentage of Th40 cells of CD3+CD4+population of mice models.

FIG. 13 is a chart that shows Th40 Cell count in peripheral blood ofApoE−/− mice

FIG. 14 is an image at 200× magnification showing Th40 cells in theshoulder region of plaque in the ApoE−/− mouse model.

FIG. 15 is a graph of interferon gamma control of Th40 proliferation.

FIG. 16 is a plot of excess interferon gamma production in CD3+CD4+CD40+cells.

FIG. 17 is a sample of stains of aortic arch of the lesser curvature ofthe aortic arch.

FIG. 18 is a stain of lesser curvature of aortic arch in control andtreated subjects.

FIG. 19 is a chart of area measurements of the lesser curvature ofaortic arches and plaques.

FIG. 20-23 are charts of treated and control ApoE mice subjects.

FIG. 23 is a chart of the LDL Cholesterol level in untreated and treatedmice.

FIG. 24 is a table of blood clot data in human subjects.

FIG. 25 is a table providing the relative peptide stability assessed byExPaSY analysis.

FIG. 26 is a western blot comparing control and treated samples fromsubject mice.

FIG. 27 is a graph of LDL cholesterol measured in treated and untreatedsubjects.

FIG. 28A is an image of KGYY6 treated aortic en-face Sudan IV staining.

FIG. 28B is an image of control aortic en-face Sudan IV staining.

FIG. 29 is a graph demonstrating the reduction of lesion areas of SudanIV staining.

FIG. 30 is a graph of plaque volume reduction for area under the curve.

FIG. 31 is a graph of plaque composition for KGYY6 treated and controlsubject mice.

FIG. 32A is an image of trichrome stained sections of KGYY6 treatedsubject.

FIG. 32B is an image of trichrome stained sections of control subjects.

FIG. 33A is a dot-plot of flow cytometry data of T1D diabetes patientswith high calcium scores.

FIG. 33B is a dot-plot of flow cytometry data of T1D diabetes with lowcalcium scores.

FIG. 34A is a chart of calcium scores versus percent Th40 cells indifferent patient populations.

FIG. 34B is a chart of calcium scores versus percent Th40 cells indifferent patient populations.

FIG. 35 is a graph of percentage of Th40 cells in low coronary arterycalcium (CAC) and high coronary artery calcium (CAC) subjects.

FIG. 36 is a graph of the percentage of interferon gamma (IFNγ) in Th40cells in low coronary artery calcium (CAC) and high coronary arterycalcium (CAC) subjects.

FIG. 37 is a graph of the percentage change of in-vitro lymphocytecytokines measured in spleen cells from ApoE−/− and C57BL/6 mice asmeasured by flow cytometry.

DETAILED DESCRIPTION

The present subject matter is based on the discovery that a uniquesubset of T-cells, which express CD40 protein, and thus are referred toas Th40 cells, that may be instrumental in autoimmune inflammation.Moreover, involvement of Th40 cells in the autoimmune process may bedependent on the interaction between CD40 protein expressed on thesurface of the T-cell, and CD154 protein. Interaction of CD40 and CD154results in activation signals being delivered between the cells, andsubsequent activation of the Th40 cell. Such activation results inpropagation of the Th40 cell and an increase in inflammation (e.g., anincrease in the number of immune cells and immunoregulatory molecules,present in the system). Accordingly, inhibition of the CD40/CD154interaction can modulate Th40 cell activity, and thereby affectinflammation. Thus the present subject matter relates to the peptides,and administration thereof, that may affect the interaction between aCD40 protein and a CD154 protein, thereby modulating inflammation.Moreover, the present subject matter relates to peptides that affect theinteraction between CD40 protein expressed on the surface of a T-cell,and a CD154 protein, thereby affecting T-cell activity, controllinginflammation, and consequently preventing, modulating, and reducingatherosclerosis. The present subject matter also encompasses the use ofsuch peptides to detect Th40 cells.

Before the present development is further described, it is to beunderstood that this invention is not strictly limited to particularembodiments described, as such may of course vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present invention will be limited onlyby the claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. It should further be understood thatas used herein, the term “a” entity or “an” entity refers to one or moreof that entity. For example, a nucleic acid molecule refers to one ormore nucleic acid molecules. As such, the terms “a”, “an”, “one or more”and “at least one” can be used interchangeably. Similarly the terms“comprising”, “including” and “having” can be used interchangeably.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited. The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates, which may need to be independently confirmed.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodiments arespecifically embraced by the present invention and are disclosed hereinjust as if each and every combination was individually and explicitlydisclosed. In addition, all sub-combinations are also specificallyembraced by the present invention and are disclosed herein just as ifeach and every such sub-combination was individually and explicitlydisclosed herein.

It is further noted that the claims may be drafted to exclude anyoptional element. As such, this statement is intended to serve asantecedent basis for use of such exclusive terminology as “solely,”“only” and the like in connection with the recitation of claim elements,or use of a “negative” limitation.

Furthermore, as used herein the term animal refers to a vertebrate,preferably a mammal, more preferably a human. Suitable mammals on whichto use the methods of the present invention include but are not limitedfarm animals, sports animals, pets, primates, mice, rats, horses, dogs,cats, and humans. The term animal can be used interchangeably with theterms subject or patient.

One embodiment of the present subject matter is a peptide that interactswith a CD40 protein in such a manner as to prevent atherosclerosis. Asused herein, the terms interact, interaction, and the like, mean thattwo molecules come into sufficient physical proximity such that theycause a modulation of inflammation. One such type of interaction is abinding interaction. In such an interaction the peptide associates withCD40 to form a complex. An example of complex formation is theassociation of an antigen with an antibody. According to the presentsubject matter, binding of a peptide hereof to a CD40 protein can bereversible (e.g., non-covalent binding interactions) or non-reversible(e.g., covalent binding interactions). Moreover, a reversibleinteraction can be strong or weak, the strength of the interaction beingdetermined by the forces (e.g., ionic charges, hydrogen binding, van derWalls interactions, etc.) exerted by each protein on the other proteinin the complex. Factors affecting the strength of an interaction betweentwo molecules are known to those skilled in the art. One useful measureof the strength of binding between two molecules, such as a peptide anda protein, is the dissociation constant (Kd). Preferred peptides of thepresent invention are those that bind to a CD40 protein with a Kd of nomore than about 1×10⁻⁶ M, about 1×10⁻⁷ M, or about 1×10⁻⁸ M.Particularly preferred peptides are those having a Kd of less than about1×10⁻⁹ M. In one embodiment, a peptide hereof binds to a CD40 proteinwith a Kd of less than 100 nM, less than 50 nM, less than 25 nM, lessthan 10 nM, less than 5 nM, less than 3 nM, less than 2 nM, or less than1 nM. Methods of measuring and analyzing binding interactions between apeptide and a CD40 protein are known by those of skill in the art.

As used herein, to modulate inflammation means to change the level ofTh40 cells present in an animal, or in a culture of T cells. As usedherein, the terms level, number, count and concentration can be usedinterchangeably. Modulation of inflammation can mean an increase ordecrease in the number of Th40 cells present in the inflammatoryenvironment. Consequently, modulation can be referred to as positive ornegative. Positive modulation (also referred to as up-regulation) ofinflammation refers to an increase in the number of Th40 cells in theinflammatory environment. Negative modulation (also referred to asdown-regulation) of inflammation refers to a reduction in the number ofTh40 cells present in the inflammatory environment. A preferred peptideis one that down-regulates inflammation, thereby reducing the number ofTh40 cells present in the inflammatory environment. Positive andnegative modulation of inflammation may or may not result in a change inthe type and amount of immunoregulatory molecules present in theinflammatory environment.

It will be appreciated by those skilled in the art that both a cellculture system and the immune system of an animal comprise basal levelsof immune cells and immunoregulatory molecules. The phrases basal leveland normal level can be used interchangeably. With regard to the immunesystem of an animal, as used herein, the basal level of a type of immunecell (e.g., Th40 cell), or a immunoregulatory molecule, refers to theaverage number of that cell type, or immunoregulatory molecule, presentin a population of individuals considered healthy (i.e., free ofmetabolic, autoimmune, or infectious disease). With regard to a cellculture system, as used herein, the basal level of a type of immunecell, or an immunoregulatory molecule, refers to the average level ofthat cell type, or immunoregulatory molecule, present in a population ofcells that is non-activated. Those skilled in the art are capable ofdetermining if a T-cell, or a population of such cells, is activated.For example, the expression of CD69, CD25 and/or CD154 proteins by acell indicates that the cell has been activated.

The basal level of a cell or molecule can be a specific amount (e.g., aspecific concentration) or it can encompass a range of amounts. Basallevels, or ranges, of immune cells and immunoregulatory molecules areknown to those in the art. For example, in a healthy individual, thenormal level of CD4+ T-cells present in human blood is 500-1500cells/ml. Variability in this measurement can result from differences inthe method used to determine the cell count. Furthermore, normal levelsof cells can also be reported as a percentage of a total cellpopulation. For example, in a healthy individual, Th40 cells make upless than 25% of the total T cell population. Thus, as used herein, theterm inflammation refers to an inflammatory environment in which Th40cells make up greater than about 25%, greater than about 30%, greaterthan about 35%, greater than about 40%, greater than about 45% , greaterthan about 50%, greater than about 55%, greater than about 60%, greaterthan about 65%, greater than about 70%, greater than about 75%, orgreater than about 80% of the total T-cell population. Moreover, apreferred peptide herein is one that reduces the level of Th40 cells toless than about 50%, less than about 45%, less than about 40%, less thanabout 35%, less than about 30%, less than about 27%, or equal to about25% of the total T-cell population. Methods of measuring different typesof T-cells in the T-cell population are known to those skilled in theart. Furthermore, a novel method for detecting Th40 cells using peptideshereof is disclosed herein.

As used herein, the phrase inflammatory environment refers to theoverall population of immune cells, and related immunoregulatorymolecules, that are present in a culture of cells, or in the body of ananimal. As such, the phrase inflammatory environment encompasses thetypes, and/or the relative amounts of immune cells and immunoregulatorymolecules (e.g., cytokines) present in a culture of cells, or in ananimal, which are involved in affecting an inflammatory reaction.Examples of cells encompassed by the term inflammatory environmentinclude, but are not limited to, T cells, neutrophils, macrophages,granulocytes, and the like. The inflammatory environment relates tocells and molecules that mediate both acute and chronic inflammation. Itwill be appreciated by those skilled in the art that the inflammatoryenvironment refers to the system to which peptides hereof areadministered. In one embodiment, the system is a cell culture system. Inone embodiment, the system is a whole animal.

A preferred peptide hereof is one that selectively interacts with a CD40protein in solution, as determined using an assay such as animmunosorbent assay, or on the surface of a T-cell. As used herein, theterms selectively, selective, specific, and the like, indicate thepeptide has a greater affinity for a CD40 protein than it does forproteins unrelated to the CD40 protein. More specifically, the termsselectively, selective, specific, and the like indicate that theaffinity of the peptide for CD40 is statistically significantly higherthan its affinity for a negative control (e.g., an unrelated proteinsuch as albumin) as measured using a standard assay (e.g., ELISA).Suitable techniques for assaying the ability of a peptide to selectivelyinteract with a CD40 protein are known to those skilled in the art. Suchassays can be in vitro or in vivo assays. Examples of useful assaysinclude, but are not limited to, an enzyme-linked immunoassay, acompetitive enzyme-linked immunoassay, a radioimmunoassay, afluorescence immunoassay, a chemiluminescent assay, a lateral flowassay, a flow-through assay, an agglutination assay, a particulate-basedassay (e.g., using particulates such as, but not limited to, magneticparticles or plastic polymers, such as latex or polystyrene beads), animmunoprecipitation assay, an immunoblot assay (e.g., a western blot), aphosphorescence assay, a flow-through assay, a chromatography assay, apolyacrylamide gel electrophoresis (PAGE)-based assay, a surface plasmonresonance assay, a spectrophotometric assay, a particulate-based assay,an electronic sensory assay and a flow cytometric assay. Methods ofperforming such assays are well known to those skilled in the art. Inone embodiment, an assay can be performed using cells in culture, or itcan be performed in a whole animal. Assays can be designed to givequalitative, quantitative or semi-quantitative results, depending on howthey are used and the type of result that is desired.

One embodiment hereof is a peptide that interacts with a CD40 protein insuch a manner as to affect the interaction of the CD40 protein with aCD154 protein, thereby modulating inflammation. The effect of thepeptide on the CD40/CD154 interaction can be positive or it can benegative. For example, the peptide can interact with the CD40 protein insuch a manner that the strength of the interaction between the CD40protein and a CD154 protein is increased. Alternatively, the peptide caninteract with the CD40 protein such that the strength of the interactionbetween the CD40 protein and a CD154 protein is decreased. Methods ofmeasuring the strength of binding between the peptide and a CD40 proteinare known to those skilled in the art. A preferred peptide hereof is onethat reduces the strength of the interaction between a CD40 protein anda CD154 protein. Preferred peptides hereof reduce the strength ofbinding between a CD40 protein and a CD154 protein by at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, or at least 95%. A particularlypreferred peptide is one that completely inhibits binding of CD40 toCD154. Complete inhibition of binding between CD40 and CD154 means thatwhen a peptide hereof is brought into proximity with a CD40 protein anda CD154 protein under conditions that would normally allow theinteraction of CD40 and CD154, no such interaction occurs and activationsignals are not stimulated in the CD40-expressing cell. ConsequentlyCD40/CD154 mediated modulation of inflammation does not occur. In oneembodiment, the peptide interacts with the CD40 protein in such a manneras to reduce the level of inflammation in the system. In one embodiment,the peptide interacts with the CD40 protein in such a manner as toinhibit the development of inflammation in the system.

While peptides hereof can interact with any site on the CD40 protein,preferred peptides interact with the CD40 protein at a location thatoverlaps with the CD154 binding site. In one embodiment, a peptidehereof interacts with the CD40 protein at the CD154 binding site. Anexample of such a peptide is a CD40 ligand competitive antagonist. Asused herein, peptides that interfere with, or inhibit, the binding of aCD154 protein to a CD40 protein are referred to as small interferingpeptides (SIPs). As used herein a small interfering peptide is a peptidethat, through physio-chemical properties, interferes with theinteraction of a CD40 protein with a CD154 protein, thereby preventingactivation signals from being delivered to the CD40-bearing cell, thuslimiting the activation of the CD40-bearing cell, and consequently,inflammation. As demonstrated herein, the consequences of suchinterference are prevention of T-cell activation and propagation, and aprevention or reduction of inflammation.

Additionally, a small interfering peptide, may, through itsphysio-chemical properties, interfere with the interaction of a CD40protein with a CD154 protein, thereby preventing activation signals frombeing delivered to the CD40-bearing cell, thus limiting the activationof the CD40-bearing cell, and consequently, modulating, inhibiting, andpreventing atherosclerosis. As demonstrated herein, the consequences ofsuch interference are prevention of T cell activation and propagation,and a prevention, reduction, or modulation of atheroscleroticdevelopments.

A peptide useful for practicing methods of the present developmentsshould be of a size sufficient to interact with CD40 protein in such amanner as to modulate atherosclerosis. It is understood by those skilledin the art that preferred peptides are relatively short since they areeasier and less expensive to produce. Preferred peptides are those thatare less than 20 amino acids in length. A preferred peptide is one thatis 4, 6, 8, 10, 13, 15, or 24 amino acids in length. In one embodiment,the peptide is an amino acid selected from the group of SEQ ID NO:3(Core-sequence see Table 1), SEQ ID NO:4 (6-mer see Table 1), SEQ IDNO:5 (8-mer mouse see Table 1), SEQ ID NO:6 (8-mer human see Table 1),SEQ ID NO:8 (15-mer human see Table 1), SEQ ID NO:9 (24-mer see Table1). The sequences of such peptides are shown below in Table 1.

TABLE 1 SEQ ID NO SEQUENCE Description  1MIETYSQPSP RSVATGLPAS MKIFMYLLTV SwissPro 27548.2FLITQMIGSV LFAVYLHRRL DKVEEEVNLH Mouse CD40 LigandEDFVFIKKLK RCNKGEGSLS LLNCEEMRRQ (CD154 Protein)FEDLVKDITL NKEEKKENSF EMQRGDEDPQ IAAHVVSEAN SNAASVLQWA KKGYYTMKSNLVMLENGKQL TVKREGLYYV YTQVTFCSNR EPSSQRPFIV GLWLKPSSGS ERILLKAANTHSSSQLCEQQ SVHLGGVFEL QAGASVFVNV TEASQVIHRV GFSSFGLLKL  2MIETYNQTSP RSAATGLPIS MKIFMYLLTV SwissPro 29965FLITQMIGSA LFAVYLHRRL DKIEDERNLH Human CD40 LigandEDFVFMKTIQ RCNTGERSLS LLNCEEIKSQ (CD154 Protein)FEGFVKDIML NKEETKKENS FEMQKGDQNP QIAAHVISEA SSKTTSVLQW AEKGYYTMSNNLVTLENGKQ LTVKRQGLYY IYAQVTFCSN REASSQAPFI ASLCLKSPGR FERILLRAANTHSSAKPCGQ QSIHLGGVFE LQPGASVFVN VTDPSQVSHG TGFTSFGLLK L  3 KGYYCore-sequence  4 KKGYYT 6-mer  5 AKKGYYTM 8-mer-mouse  6 AEKGYYTM8-mer human  7 VLQWAKKGYYTMKSN 15-mer-mouse  8 VLQWAEKGYYTMSNN15-mer human  9 NAASVLQWAKKGYYTMKSNLVMLE 24-mer 10 ISQAVHAAHAEINEAGR15-mer from ovalbumin; control peptide 11 G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-NGly-1 12 V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-2 13V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 14 V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-NGly-4 15 V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-5 16V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-6 17 V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-NGly-7 18 V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N Gly-8 19V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N Gly-9 20 V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-NGly-10 21 V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N Gly-11 22 ISQAVHAAHAEINEAGR15-mer from ovalbumin; control peptide 23 YVQGKANLKSKLMYTScrambled peptide 24 WAKKGYYTMK 10-mer mouse 25 VLQWAKKGYYTMK13-mer mouse 26 AASVLQW AKKGYYTMKSNLVVLEN 24-mer mouse 27 KGYYTM6-mer (Form 2) 28 AEKGYY 6-mer (Form 3) 29 AKKGYY 6-mer (Form 4) 30AKGYYT 6-mer (Form 5) 31 YKNVKQMAYWLTGKS Scrambled peptide 32AASVLQWAKKGYYTMKSNLVMLEN 24-mer-mouse (Form 2)

Interaction of a CD40 protein and a CD154 protein has been shown tooccur at particular regions within each protein. The inventors have nowshown that, surprisingly, a peptide comprising only a short portion ofthe CD154 region that interacts with CD40 is capable of binding to aCD40 protein, thereby modulating atherosclerosis. Thus one embodimenthereof is a peptide that comprises at least a portion of the amino acidsequence of a CD154 protein such that the peptide interacts with CD40protein in such a manner as to modulate atherosclerosis. In oneembodiment, interaction of the peptide with CD40 protein results innegative modulation of atherosclerosis. In one aspect, the peptidecomprises at least a portion of SEQ ID NO:1 or SEQ ID NO:2.

In one aspect, the peptide is as short as possible yet comprises enoughof the CD154 protein to allow interaction with a CD 40 protein in such amanner as to modulate atherosclerosis. In one embodiment, a peptidehereof comprises 6, 13 or 15 contiguous amino acids from SEQ ID NO:1 orSEQ ID NO:2, and interacts with CD40 in such a manner as to modulateatherosclerosis. A preferred peptide comprises a core sequence oflysine-glycine-tyrosine-tyrosine (KGYY; SEQ ID NO:3), which correspondsto amino acids 142-145 of SEQ ID NO:1 and amino acids 143-146 of SEQ IDNO:2. Useful peptides can comprise additional regions of sequence fromSEQ ID NO:1 or SEQ ID NO:2 that are adjacent to the core sequence, solong as the peptide is capable of modulating atherosclerosis. In oneembodiment hereof, a peptide comprises at least one sequence selectedfrom SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQID NO:9 and SEQ ID NO:10,so long as the peptide interacts with CD40 protein in such a manner asto modulate atherosclerosis. In one embodiment of the present subjectmatter, a peptide hereof is a sequence selected from SEQ ID NO:4, SEQ IDNO:8, SEQID NO:9 and SEQ ID NO:10.

While peptides of the present subject matter can be selected entirely ofor from sequences that are responsible for the interaction of thepeptide with a CD40 protein, they may additionally contain amino acidsequences that do not interact with a CD40 protein, but which have otheruseful functions. Any useful, additional amino acid sequence can beadded to the CD40-interacting sequence, so long as the additionalsequences do not have an unwanted affect on the ability of the CD40interacting sequence to interact with a CD40 protein. For example, inaddition to the amino acid sequence responsible for interacting with aCD40 protein, a peptide hereof can contain amino acid sequences that areuseful for visualizing or purifying the peptide. Such sequences act aslabels (e.g., enzymes) or tags (antibody binding sites). Examples ofsuch labels and tags include, but are not limited to, B-galactosidase,luciferase, glutathione-s-transferase, thioredoxin, HIS-tags, biotintags, and fluorescent tags. Other useful sequences for labeling andtagging proteins are known to those of skill in the art.

Likewise, peptides hereof can be modified, so long as such modificationdoes not significantly affect the ability of the peptide to modulateatherosclerosis. Such modifications can be made, for example, toincrease the stability, solubility or absorbability of the protein.Examples of such modifications include, but are not limited topegylation, glycosylation and chemical modification of the peptide.

Peptides hereof can be obtained from nature (e.g., obtained from plants,animals or microorganisms) or they can be produced in a laboratory(e.g., recombinantly or synthetically). Preferred peptides are thosethat are synthesized. Also encompassed are peptides that arecombinations of natural and synthetic molecules. General methods forproducing and isolating recombinant or synthetic peptides are known tothose skilled in the art. It should be noted that, as used herein, anisolated, or biologically pure, molecule, is one that has been removedfrom its natural milieu. As such the terms isolated, biologically pure,and the like, do not necessarily reflect the extent to which the proteinhas been purified. As has been described herein, interaction of the CD40protein and the CD154 protein are necessary for involvement of Th40cells in atherosclerosis. Consequently, inhibition of the interactionbetween a CD40 and CD154 protein using peptides hereof is a usefulmethod of affecting atherosclerosis. Thus one embodiment is a method toreduce the interaction between a CD40 protein and a CD154 proteincomprising introducing into an environment containing a CD40 protein anda CD154 protein, a peptide, that interacts with the CD40 protein in sucha manner as to reduce the interaction between the CD40 protein and theCD154 protein. In one aspect hereof, the peptide reduces the interactionbetween the CD40 protein and the CD154 protein by at least 5%, at least10%, at least 20%, at least 30%, at least 40%, at least 50%, at least60%, at least 70%, at least 80%, at least 90%, or at least 95%. In oneembodiment, the peptide reduces the interaction between the CD40 proteinand the CD154 protein by a factor of at least 10, at least 100, at least1,000, at least 10,000. Methods of measuring the strength of theinteraction between the CD40 protein and the CD154 protein have beendiscussed previously, and are also know to those of skill in the art.

One embodiment hereof is a method to modulate atherosclerosis comprisingcontacting a CD40 protein with a peptide that interacts to the CD40protein in such a manner as to modulate inflammation. In one aspect,interaction of the peptide with the CD40 protein increases the number ofTh40 cells by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95%. In one embodiment, interaction of thepeptide with the CD40 protein increases the number of Th40 cells by afactor of at least 10, at least 100, at least 1,000, at least 10,000.

One aspect is a method to reduce atherosclerosis in a patient, themethod comprising administering a peptide hereof to the patient. In oneembodiment, the peptide comprises an amino acid sequence selected fromSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. Inone embodiment, the peptide is an amino acid sequence selected from SEQID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. In a preferredembodiment, interaction of the peptide with the CD40 protein decreasesthe number of Th40 cells by at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%. In another embodiment,interaction of the peptide with the CD40 protein decreases the number ofTh40 cells by a factor of at least 10, at least 100, at least 1,000, atleast 10,000. In a preferred embodiment, the level of Th40 cells isreduced so that Th40 cells comprise no more than about 20%, about 25%,about 30%, about 35%, or about 40% of the total T-cell population.

Peptides and methods hereof are suitable for use in cell culture as wellas for treating a patient. As used herein the term patient refers to anyanimal in need of such treatment. The animal can be a human or anon-human animal. A preferred animal to treat is a mammal. A peptide canbe administered or applied per se, or as pharmaceutical compositions. Apeptide hereof, or a pharmaceutical composition thereof, can beadministered to a patient by a variety of routes, including, but limitedto, by injection (e.g., intravenous, intramuscular, subcutaneous,intrathecal, intraperitoneal), by inhalation, by oral (e.g., in a pill,tablet, capsule, powder, syrup, solution, suspension, thin film,dispersion or emulsion.), transdermal, transmucosal, pulmonary, buccal,intranasal, sublingual, intracerebral, intravaginal rectal or topicaladministration or by any other convenient method known to those of skillin the art.

The amount of a peptide hereof and/or a pharmaceutical compositionthereof that will be effective can be determined by standard clinicaltechniques known in the art. Such an amount is dependent on, among otherfactors, the patient being treated, including, but not limited to theweight, age, and condition of the patient, the intended effect of thecompound, the manner of administration and the judgment of theprescribing physician. Also, in this context, it should be noted that intreating a patient exhibiting a disorder of interest, a therapeuticallyeffective amount of an agent or agents such as these is administered. Atherapeutically effective dose refers to that amount of the compoundthat results in amelioration of one or more symptoms or a prolongationof survival in a patient.

A peptide hereof, or a pharmaceutical composition thereof, can beadministered alone or in combination with one or more otherpharmaceutical agents, including other compounds of the presentdisclosure. The specific pharmaceutical composition depends on thedesired mode of administration, as is well known to the skilled artisan.

Because the inventors have discovered that Th40 cells are intimatelyinvolved in the development of autoimmune diseases and atherosclerosis,the peptides and methods disclosed herein can be used to affectatherosclerosis resulting from such diseases. Thus, one embodimenthereof is a method to treat atherosclerotic disease in a patient in needof such treatment, the method comprising administering to a patient apeptide that interacts with the CD40 protein, thereby reducingatherosclerosis. In one embodiment the peptide interacts with the CD40protein in such a manner as to affect the interaction of CD40 and CD154,thereby reducing atherosclerosis. In a preferred embodiment, interactionof the peptide with the CD40 protein reduces the number of Th40 cells ina patient to a level equal to that observed in subjects that do not havecardiovascular disease. The present developments are suitable fortreating any patient having an autoimmune disease and/or cardiovasculardisease, the development of which is dependent on Th40 cells. Morespecifically, peptides hereof are suitable for reducing the level ofTh40 cells in such patients. In a preferred embodiment, a peptide hereofreduces the level of Th40 cells in a patient suffering from acardiovascular disease to no more than about 25% of the total T-cellpopulation.

One example of a disease that is particularly amenable to treatmentusing a peptide of the present developments may be atherosclerosis. Inatherosclerosis, inflammatory changes of the arterial wall occurresulting in the formation and buildup of arterial plaque. Consequently,control of inflammatory cells and cell signaling via CD40-CD154interaction may be able to be used to control, modulate, and/or reduceatherosclerotic lesions that are characterized as chronicinflammatory-fibroproliferative disease of the vessel wall. Severalmurine models of atherosclerosis have been developed. The progression oflesion formation is observable in apolipoprotein E (apoE) deficienttransgenic mice and can be observed by measurement of the aortic arch,the number and type of plaque, and characterized in accordance with theAmerican Heart Association's staging of atherosclerosis, ranging fromAHA type I to AHA type V. AHA type 1, may be characterized by early orinitial lesions, may be comprised of histologically “normal” cells,macrophage infiltration, and isolated foam cells. AHA type V, may becharacterized by advanced or complicated legions, including but notlimited to increased endothelial dysfunction characterized by surfacedefects, hematoma, hemorrhage, and/or thrombosis. Thus, one embodimentof the present developments is a method to prevent atherosclerosis in anindividual at risk for developing atherosclerosis, the method comprisingadministering to the individual a peptide to selectively bind to a CD40expressing cell.

The risk for atherosclerosis may result from familial factors (e.g.,inheritance) or from other factors, such as the physical condition ofthe individual. Some methods of risk assessment are known to thoseskilled in the art. In one embodiment, the peptide comprises an aminoacid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQID NO:9 and SEQ ID NO:10, so long as the peptide can down-regulateinflammation. In one embodiment, the peptide is an amino acid sequenceselected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQID NO:10.

The inventors have also shown that, surprisingly, peptides hereof can beused to reverse the disease process in individuals already showing signsof atherosclerosis. Thus, one aspect of the present subject matter is amethod to reverse atherosclerosis comprising administering to a patientdiagnosed as having atherosclerosis, a peptide hereof. In oneembodiment, the peptide comprises an amino acid sequence selected fromSEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10, solong as the peptide can down-regulate inf. In one embodiment, thepeptide is an amino acid sequence selected from SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. As used herein thephrase to reverse atherosclerosis means to reduce the aortic-archinfiltration, plaque, and lesions of an individual with atherosclerosisto a level comparable to that observed in a non-atheroscleroticindividual.

As has been described, peptides of the present development selectivelybind to a CD40 expressing cell. Consequently, peptides of the presentsubject matter can be used to identify Th40 cells. Thus one embodimenthereof is a method to detect Th40-dependent atherosclerosis, said methodcomprising contacting a T-cell population with a peptide hereof. In apreferred embodiment, the peptide is labeled with a detectable marker,such as, for example, luciferase or alkaline phosphatase. Such detectioncan be performed using assay techniques known to those skilled in theart. In general, an assay for detecting Th40 cells using a peptidehereof comprises (a) obtaining a sample of cells; (b) contacting apeptide hereof with said cells under condition suitable to allow bindingof the peptide to Th40 cells, if present; (c) washing said cells usingconditions that disrupt non-specific interactions, and that removeunbound peptide; and (d) detecting peptide bound to cells. Detection ofbound peptide can be achieved directly or indirectly. For example,direct detection can be achieved using a peptide labeled using adetectable marker, as disclosed herein. Following the wash step listedabove, the cells are then simply screened for the presence of detectablemarker. The presence of detectable marker in the cell sample indicatesthe presence of Th40 cells, and thus Th40-dependent atherosclerosis.Alternatively, indirect detection involves the use of a second molecule,such as an antibody, that binds to the peptide. In an indirect detectionassay, following the wash step listed above, a detection molecule thatbinds the peptide is added to the cell sample. The detection molecule islabeled with a detectable marker. After washing away unbound detectionmolecule, the cells are screened for the presence of detectable marker.The presence of detectable marker in the cell sample indicates thepresence of Th40 cells. It should be understood that the assaysdescribed herein are meant as examples of useful assays, and other assaytechniques can be employed. Suitable assay techniques are known to thoseskilled in the art, and are also disclosed in, for example, MolecularCloning: A Laboratory Manual, Sambrook, J., Fritsch, E. F., andManiatis, T, Cold Spring Harbor Laboratory Press; 2nd Edition (December1989). All references cited herein are incorporated herein in theirentirety.

The assay technology described above can also be used to identify othermolecules that affect the interaction of a CD40 protein with a CD514protein. Examples of such molecules include, but are not limited to,proteins, peptides and small molecules. For example, assays can bedesigned that test the ability of molecules to compete with a peptide ofthe present developments for binding to a Th40 cell. For instance, apeptide labeled with a detectable marker, can be mixed with a testmolecule and a population of cells known to contain Th40 cells, underconditions that allow binding of the peptide to the Th40 cells.Following an appropriate incubation period, the cells are washed toremove unbound peptide, and the cells screened for the presence ofdetectable marker. Alternatively, the labeled peptide could be bound toTh40 cells first, and after a wash step to remove unbound peptide, thetest molecule could be added to the cells containing bound peptide.Following an incubating period and a wash step to remove unboundmolecule, or released peptide, the cells are screened for the presenceof detectable marker. In either case, absence of the detectable markerin the cell sample indicates the test molecule is able to compete withthe peptide for binding to the Th40 cells, while presence of thedetectable marker would indicate the test molecule does not inhibitbinding of the peptide to Th40 cells. Inhibition of binding need not be100%, as such assay would also be useful for identifying molecules thatpartially inhibit binding of the peptide to Th40 cells. It is understoodby those skilled in the art that such assays would involve the use ofpositive controls (e.g., unlabeled peptide) and negative controls (e.g.,a protein/molecule that is known not to bind to Th40 cells).

Because increased levels of Th40 cells are associated with thedevelopment of autoimmune disease, the present developments can be usedto identify patients at risk for developing autoimmune disease andautoimmune related atherosclerosis and/or cardiovascular disease moregenerally. Thus, one embodiment of the present developments is a methodto identify a patient at risk for developing autoimmune relatedatherosclerosis. In one embodiment, patients at risk for developingatherosclerosis are identified by obtaining a sample from a patient tobe tested, contacting the T-cell portion of said sample with a peptidehereof, and determining the level of Th40 cells present in the sample,wherein a level of Th40 cells greater than about 25% of the total T-cellpopulation indicates the patient is at risk for developingatherosclerotic disease. In one embodiment, the peptide comprises anamino acid sequence selected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8,SEQ ID NO:9 and SEQ ID NO:10, so long as the peptide binds to the CD40protein. In one embodiment, the peptide is an amino acid sequenceselected from SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQID NO:10. In a preferred embodiment the peptide is labeled with asuitable detectable marker such as, for example, luciferase or alkalinephosphatase.

The present developments also comprise kits useful for practicing themethods disclosed herein, the kit comprising a peptide that interactswith a CD40 protein in such a manner as to modulate atherosclerosis. Inone embodiment, the peptide comprises an amino acid sequence selectedfrom SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ IDNO:10, so long as the peptide can down-regulate atherosclerosis. In oneembodiment, the peptide is an amino acid sequence selected from SEQ IDNO:3, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:9 and SEQ ID NO:10. Anotherembodiment is a kit for determining the level of Th40 cells, the kitcomprising a peptide that interacts with a CD40 protein, and methods fordetecting CD40-bound peptide. Kits can also contain associated reagentsand components, such as, but not limited to, buffers, labels,containers, inserts, tubing, vials, syringes, and the like.

The present developments also comprise a kit or method useful forpracticing the methods disclosed herein, the kit or method comprising amethod to confirm or rule out diagnosis of cardiovascular disease,atherosclerosis, and/or coronary artery disease in a patient comprising:determining the percentage of Th40 cells in a sample isolated from asubject presenting with Type 1 Diabetes, comparing the percentage ofTh40 cells to a control sample or a standard value, and, diagnosingcardiovascular disease and/or coronary artery disease in the subjecthaving an increase in the percentage of Th40 cells in the sample fromthe subject relative to the control sample or standard value; or, rulingout cardiovascular disease, atherosclerosis, and/or coronary arterydisease in the subject having no increase or a decrease in thepercentage of Th40 cells in the sample from the subject relative to thecontrol sample or standard value.

The present developments also comprise a kit or method useful forpracticing the methods disclosed herein, the kit or method comprising amethod to confirm or rule out diagnosis of cardiovascular disease,atherosclerosis, and/or coronary artery disease in a patient comprising:determining the percentage of Th40 cells in a sample isolated from asubject presenting with symptoms suggestive of cardiovascular disease,atherosclerosis, and/or coronary artery disease, comparing thepercentage of Th40 cells to a control sample or a standard value, anddiagnosing cardiovascular disease, atherosclerosis, and/or coronaryartery disease in the subject having an increase in the percentage ofTh40 cells in the sample from the subject relative to the control sampleor standard value is indicative of cardiovascular disease,atherosclerosis, and/or coronary artery disease in the subject.

Another embodiment of the developments hereof is a method to confirm orrule out diagnosis of cardiovascular disease, atherosclerosis, and/orcoronary artery disease in a patient comprising: determining thepercentage of Th40 cells in a sample isolated from a subject,determining the percentage of interferon gamma (IFNγ) in a sampleisolated from a subject, comparing the percentage of Th40 cells to acontrol sample or a standard value, comparing the percentage ofinterferon gamma (IFNγ) to a control sample or standard value, anddiagnosing cardiovascular disease and/or coronary artery disease in thesubject having an increase in the percentage of Th40 cells andinterferon gamma (IFNγ) in the sample from the subject relative to thecontrol sample or standard value; or, ruling out cardiovascular disease,atherosclerosis, and/or coronary artery disease in the subject having noincrease or a decrease in the percentage of Th40 cells and interferongamma (IFNγ) in the sample from the subject relative to the controlsample or standard value.

The present developments also comprise a kit or method useful forpracticing the methods disclosed herein, the kit or method comprising amethod for identifying and treating a patient for cardiovasculardisease, the method comprising detecting the percentage of Th40 cells inT-cells in a blood sample from the patient and treating the patient withelevated percentage of Th40 cells relative to a percentage of Th40 cellsin a blood sample from a normal control sample with a drug in an amountto decrease the percentage of Th40 cells in the patient's blood.

The following examples are provided for the purpose of illustration andare not intended to limit the scope of the present invention.

EXAMPLES Example 1

This Example demonstrates the effect of various peptide fragments ofCD154 on CD4/CD8 ratios and the development of diabetes in NOD mice.

Peptides were designed based on the amino acid sequence of mouse CD154protein (SEQ ID NO:1) in the SwissPro database. The peptides (8-mer (SEQID NO: 5; SEQ ID NO: 6), 10-mer (SEQ ID NO:24), 13-mer (SEQ ID NO:25),15-mer (SEQ ID NO: 7), 24-mer (SEQ ID NO:26), scrambled (SEQ ID NO: 23),and RGD (arginylglycylaspartic acid) were then ordered from New EnglandPeptide. The RGD peptide is a 15-amino acid sequence from the CD154sequence that does not include the CD40 binding motif. The lyophilizedpeptides were suspended in sterile saline at 1 mg/ml. 25 ug (1 mg/kg) ofa particular peptide was then injected into the tail vein of 6-week oldNOD mice. Control mice received 100 ul of sterile saline. This is wellbefore the onset of diabetes (and atherosclerosis), but after damage topancreatic islets has begun. Weekly after the initial injection, another100 ug of peptide (or 100 ul of saline in the case of the Control mice)was injected into the tail vein. At 10 weeks of age, mice were monitoredfor diabetes, as indicated by a blood glucose level greater than 250mg/dL for three consecutive days. The results of this study are shown inFIG. 1. During this time, blood was also taken from the tail vein, or bysub-mandibular venal puncture, and the level of CD4+ and CD8+ cellsdetermined by flow cytometry using antibodies for CD4 protein and CD8protein. The results of this analysis are shown in FIG. 2A.

Pancreata were excised and examined by histology for cellularinfiltrates and assigned scores based on observable, measurable, andquantifiable data: 0=no infiltrate; 1=one pole infiltrate;2=peri-insulitis, bi-polar-infiltrates; 3=75% infiltrate and 4=fullinfiltration. The results of this analysis are shown in FIG. 2B.

The results demonstrate that treatment with a peptide unrelated to theCD154 protein did not reduce the development of diabetes in NOD mice. Incontrast, treatment of mice with a 15-mer peptide derived from the CD154protein prevented the onset of diabetes. Further, the 13-mer peptidesderived from the CD154 protein had significant effects on thedevelopment of diabetes. In addition, the data demonstrates that the15-mer peptide did not result in compromise of the immune system, asdetermined by the CD4/CD8 ratio.

Example 2

This Example demonstrates the effect of the 15-mer peptide onhyperglycemia in newly diabetic NOD mice.

Six mice from that had received the 6-mer peptide in Example 1, and thathad subsequently developed diabetes, were injected intravenously with100 ug of the 15-mer peptide. These mice were then given weeklyinjections of the 15-mer peptide into their tail veins, and their bloodglucose levels monitored twice-weekly. The 15-mer peptide wasadministered for a total of ten weeks, after which the treatment wasstopped. The results of this study are shown in FIG. 3.

This study demonstrates that injection of the 15-mer peptide intoalready diabetic mice can reverse hyperglycemia. It also demonstratesthat cessation of the treatment results in return of hyperglycemiawithin 7 weeks.

Example 3

This study demonstrates the ability of the 15-mer peptide to bind toTh40 cell and B cells.

Total lymphocytes were isolated from 9 week old NOD mice. Thelymphocytes were incubated with anti-CD, anti-CD8, and an FITC-labeled15-mer peptide, and then analyzed by flow cytometry. Cells were gatedfor CD4 (both CD4hi and CD4lo populations were included) and CD4 versusthe 15-mer peptide. The results of this analysis are shown in FIG. 4.

B cells were isolated from the spleens of NOD mice. Sorted MHC-II+ cellswere purified from total lymphocytes. Cells were stained withFITC-labeled 15 mer peptide, anti-CD40, and B cell markers CD19 andCD21. MHC-II+ cells were gated for CD19+ and CD21+ and then 15-merpeptide versus CD40 antibody was measured. The results of this study areshown in FIG. 5.

This study shows that a substantial majority, 90% of CD40+ T-cells, alsobind the 15-mer peptide, thereby demonstrating that the 15-mer peptideis highly specific for CD40+ cells. It also shows that while 90% of Bcells were CD40 positive, only 8% of B cells bound the 15-mer peptide.

Example 4

This example demonstrates the level of CD40 positive cells in the bloodof type-I diabetic subjects and non-diabetic (control) subjects.

1 ml of whole blood was obtained from each individual and incubated withbiotin-conjugated, 15-mer peptide. The cells were then exposed tohorseradish peroxidase (HRP)-avidin, washed and the absorbance at 405 nmdetermined using a spectrophotometer. The results of this study areshown in FIG. 6.

This study demonstrates that blood cells from patients having type-Idiabetes had higher 15-mer peptide binding activity than cells fromnon-diabetic controls.

Example 5

This example demonstrates the level of insulin granulation observed inthe pancreas of NOD mice treated with either the 15-mer peptide or apeptide from ovalbumin.

At the onset of diabetes, six NOD mice were injected with 100 ug/ml ofthe 15-mer peptide (SEQ ID NO:9), resulting in the reversal ofhyperglycemia in 80% of the recipients. Six weeks after reversal ofhyperglycemia, mice were sacrificed and the pancreas removed foranalysis. The pancreas was fixed, sectioned and then stained using analdehyde/fuschsin stain that allows detection of insulin granules.Granulation of the tissue was scored as follows: 4=completelygranulated; 3=75% of islet granulated; 2=50% of islet granulated, andperi-insulitis; 1=25% of islet granulated; 0=no insulin granulesdetected. The results of this analysis are shown in FIG. 7.

This analysis demonstrates that the 15-mer peptide preserved insulingranules in the majority of the mice, and was significantly improved inpeptide-reversed diabetic mice compared to diabetic mice that receivedan irrelevant peptide.

Example 6

This example demonstrates the effect of mutations in the 15-mer peptideon its ability to prevent the onset of diabetes.

Peptide were designed and produced as described in Example 1. Variantpeptides were produced so that in each variant, a glycine wassubstituted for an amino acid corresponding to an amino acid inpositions 1-9 of SEQ ID NO:9, as follows:

Gly-1 (SEQ ID NO: 11) G-L-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-2(SEQ ID NO: 12) V-G-Q-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-3 (SEQ ID NO: 13)V-L-G-W-A-K-K-G-Y-Y-T-M-K-S-N Gly-4 (SEQ ID NO: 14)V-L-Q-G-A-K-K-G-Y-Y-T-M-K-S-N Gly-5 (SEQ ID NO: 15)V-L-Q-W-G-K-K-G-Y-Y-T-M-K-S-N Gly-6 (SEQ ID NO: 16)V-L-Q-W-A-G-K-G-Y-Y-T-M-K-S-N Gly-7 (SEQ ID NO: 17)V-L-Q-W-A-K-G-G-Y-Y-T-M-K-S-N Gly-9 (SEQ ID NO: 18)V-L-Q-W-A-K-K-G-G-Y-T-M-K-S-N Gly-10 (SEQ ID NO: 19)V-L-Q-W-A-K-K-G-Y-G-T-M-K-S-N Gly-11 (SEQ ID NO: 20)V-L-Q-W-A-K-K-G-Y-Y-G-M-K-S-N Gly-12 (SEQ ID NO: 21)V-L-Q-W-A-K-K-G-Y-Y-T-G-K-S-N

NOD mice were placed in groups of 10, and the mice in each groupinjected IV weekly with 50 ug of either wild-type (WT; Legend) peptideor a variant peptide (in PBD, ph 7.2) listed above. The development ofdiabetes was monitored by measuring blood glucose levels on a weeklybasis. Mice were considered “diabetic” when blood glucose was 250 mg/dlor greater for 2 consecutive readings. Injections began at 6 weeks ofage=pre-diabetes.

This example demonstrates that substitution of a glycine at any ofpositions 1-7, or 9-12, reduces the ability of the 15-mer peptide toinhibit the development of diabetes. It also shows that such mutationsdo not completely abolish the ability of the mutated 15-mer peptide toinhibit the development of diabetes.

Example 7

This example demonstrates that the same elevation of Th40 cell levels inthe ApoE deficient mouse model of atherosclerosis is also notablyelevated in human Type 1 Diabetes (T1D).

The peripheral blood was measured was measured for total count ofCD3+CD4+CD40+ cell numbers in NOD, NOR (non-obese diabetic resistant),and BALB/c (control) mice as in FIG. 9. This was compared to thepercentage of Th40 cells in peripheral blood in human subjects forcontrol, diabetic/new onset, and long term diabetic populations as inFIG. 10. Further, lymphocytes were isolated from 9-week old NOD mice.The lymphocytes were incubated with anti-CD, anti-CD8, and anFITC-labeled 15-mer peptide, and then analyzed by flow cytometry. Cellswere gated for CD4 (both CD4hi and CD4lo populations were included) andCD4 versus the 15-mer peptide. These results are displayed in FIG. 11.

ApoE deficient mice on a normal chow diet were selected to receive adose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by IV tailinjection, three times a week over a period of 26 weeks, beginning at 9weeks of age and also utilized a control. At 25 weeks, the animals wereeuthanized, weighed, and then had blood, spleen, and pancreas removedfor analysis. The subjects were then perfused through cardiac puncturewith 4% paraformaldehyde. Aortic arches were dissected, dehydrated insucrose gradient and then flash frozen. Approximately thirty-five Bumlongitudinal sections were obtained per mouse for various stainingprocedures. Flow cytometry was performed utilizing a MACSQuant® Analyzer10 (Miltenyi Biotec Inc.). Additional analysis was performed usingFlowJo® (FlowJo, LLC wholly owned by BectonDickinson, Inc.) single-cellflow cytometry software.

NOD mice demonstrated increased levels of Th40 cells relative to allCD3+CD4+ cells prior to hyperglycemia as demonstrated in FIG. 12.

Further, NOD mice tested in this study demonstrated significant Th40infiltration in the aorta compared with control and young non-diabeticNOD mice populations, as shown in FIG. 13.

An exemplar aortic plaque of one of the longitudinal sections wasobserved at 200× magnification using oil-red-0, trichrome stain andimmune-fluorescence, and is shown in FIG. 14. This microscopy and stainof the aorta showed that not only are the Th40 cells increased in theaorta similarly to the peripheral blood as demonstrated in FIGS. 10-14,but also the Th40 cells are found within the shoulder region of plaquein the ApoE−/−model (the growth region of plaque/atherosclerosis). InFIGS. 14, 10 and 20 identify cells that represent CD3+, CD4+, and CD40+(Th40 cells) that have significant intracellular CD40. 30 demonstratesTh40 cell with extracellular expression and no demonstrative CD40intracellularly. 40 identifies CD3+, CD4+, CD40 neg cell.

Example 8

This example demonstrates interferon gamma production in CD3+CD4+CD40+cells appear to produce interferon gamma (IFNγ) in abundance.Additionally, interferon gamma controls Th40 proliferation.

ApoE deficient mice on a normal chow diet were selected to receive adose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by IV tailinjection, three times a week over a period of 26 weeks, beginning at 9weeks of age and also utilized a control. At 25 weeks, the animals wereeuthanized, weighed, and then had blood, spleen, and pancreas removedfor analysis. The subjects were then perfused through cardiac puncturewith 4% paraformaldehyde. Aortic arches were dissected, dehydrated insucrose gradient and then flash frozen. Approximately thirty-five Bumlongitudinal sections were obtained per mouse for various stainingprocedures. Flow cytometry was performed utilizing a MACSQuant® Analyzer10 (Miltenyi Biotec Inc.). Additional analysis was performed usingFlowJo ® (FlowJo, LLC wholly owned by BectonDickinson, Inc.) single-cellflow cytometry software.

As demonstrated in FIG. 14 through confocal microscopy, CD40 can beinternal or external to the CD3+CD4+ cell. Flow cytometry was furtherperformed and demonstrated that while most CD3+ cells appear to haveability to produce CD40, the CD3+CD4+CD40+ cells appear to produceinterferon gamma (IFNγ) in abundance. This flow cytometry studyincorporated both the external and internal staining of CD3, CD4, CD40,and IFNγ.

FIG. 16 demonstrates that interferon gamma controls Th40 proliferation.Isolated Th40 cells were cross-linked by antibody to CD40. The graph inFIG. 16 denotes proliferation of cross-linked (activated) Th40 cells(CD40 XL) vs. antibody to IFNγ and non-cross linked controls (UN).Additionally, by affecting IFNγ, activated Th40 cells do notproliferate.

Example 9

This example demonstrates that KGYY₁₅ (15-mer—SEQ ID NO:7) abrogatesatherosclerosis.

ApoE deficient mice on a normal chow diet were selected to receive adose of 1 mg/kg of the 15-mer peptide (SEQ ID NO: 7) by IV tailinjection, three times a week over a period of 26 weeks, beginning at 9weeks of age and also utilized a control. At 25 weeks, the animals wereeuthanized, weighed, and then had blood, spleen, and pancreas removedfor analysis. The subjects were then perfused through cardiac puncturewith 4% paraformaldehyde. Aortic arches were dissected, dehydrated insucrose gradient and then flash frozen.

FIG. 17 provides an example of the lesser curvature of the aortic arch,defined proximally from the aortic outflow (AO). Of the segments seen inthe trichrome stain, an intimal distance of 2.4 mm was measureddistally. The aortic-arch wall area subtended by this 2.5 mm stretch ofthe intima was calculated for each section of all mice, with maximalarea of the inner-aortic-arch wall of each mouse used to computeaverages per group. The luminal surface (L), aortic arch (AO), andinnominate artery (I) are labelled in this FIG. 17.

FIG. 18 demonstrates the lesser curvature of the control ApoE micecompared to the lesser curvature of mice treated with the 15-merpeptide, in accordance with the steps outlined in this example.

FIG. 19 demonstrates the reduction of the total area achieved by peptidetreatment. The total area of the 2.5 mm segment (as described in FIG.17) was substantially reduced.

FIG. 20 demonstrates the reduction of number of plaque, including earlylesions and advanced plaque. The total number of plaque wassignificantly decreased in the treatment group. Plaque was subdividedbased on morphology of early lesions (observable as fatty streakscontaining macrophage derived foam cells with varying degrees of lipidaccumulation) compared with more advanced fibroatheromas (containingvarying degrees of lipid or necrotic core and fibrous caps). All plaquewithin the designated 2.5 mm segment were included. Both the totalnumber and type of plaque were significantly decreased in those subjectstreated with the peptide.

This study demonstrates that administration of the peptide abrogatesatherosclerosis.

Example 10

This example demonstrates administration of the KGYY₁₅ (15-mer—SEQ IDNO:7) augments cap formation while reducing advancement of existingdisease.

In this study, six ApoE−/− mice received a normal chow diet from 0 to 20weeks of age. At 20 weeks of age, three mice were randomly assigned toreceive dose of 1 mg/kg of KGYY₁₅ (15-mer—SEQ ID NO:7) by IV tailinjection, once a week for a period of 4 weeks. Control mice receivedvehicle only. After 4 weeks of treatment, animals were euthanized thenperfused through cardiac puncture with 4% paraformaldehyde. Aorticarches were dissected in surcrose gradient and flash frozen.Approximately fifty, Bum longitudinal sections were obtained per mouse.Slides were treated with trichrome stain and analyzed using cellSenssoftware for measurements. Total plaque was measured including 2.5 mmlesser curvature and innominate artery.

FIG. 20 shows the number of individual early plaques and advancedplaques were reduced in the treated subjects compared to the controlsubjects.

FIG. 21 shows that the cap to core ratio of advanced plaques was reducedin the treated subjects compared to the control subjects.

FIG. 22 shows the average cap width (cap size) was greater in thetreated subjects compared to the control subjects.

FIG. 23 shows the average core width (core size/plaque size) wasdecreased in the subjects treated with the peptide compared to thecontrol subjects.

This example demonstrates that administration of the KGYY₁₅ (15-mer—SEQID NO:7) augments cap formation while reducing advancement of existingdisease. Moreover, this study further demonstrates that administrationof the KGYY15 peptide trends toward results of plaque stability.

From the foregoing, it is readily apparent that T1D shares withatherosclerosis the CD40-CD154 dyad which drives autoimmuneinflammation. There are increased Th40 cell levels in peripheral bloodof NOD mice, human T1D patients, and ApoE−/− mice. Th40 cells infiltratethe aortic wall and are found within the plaque of ApoE−/− mice. Th40cells produce the inflammatory cytokine IFNγ at a level greater thanthat of other cells and this drives inflammation. The KGYY₁₅ peptidetargets Th40 cells. The specified peptide furthermore abrogates andmodulates atherosclerosis which may be due to Th40 interaction orgeneral interaction on CD40. Moreover, the administration of thespecified peptide trends toward more stable plaque types.

Example 11

Whole human blood was administered the peptide in accordance withsimilar dosing levels to those used for murine studies.

FIG. 24 provides the results of clot studies observed in humans.

This study demonstrates that the KGYY15 peptide when administered tohumans does not modify or change the clotting of whole human bloodsignificantly outside of normally recognized levels.

Example 12

This example demonstrates that ApoE mice that have been geneticallymodified to obtain atherosclerosis and fed a high fat diet and treatedwith the 6-mer peptide may have the levels of LDL cholesterol valuesdecreased compared to untreated subjects.

In this study, six ApoE−/− mice received a normal chow diet from 0 to 20weeks of age. At 20 weeks of age, three mice were randomly assigned toreceive dose of 1 mg/kg of KGYY₆ (6-mer—SEQ ID NO:4) by IV tailinjection, once a week for a period of 4 weeks. Control mice receivedvehicle only.

Data obtained from untreated mice and compared to those in treated miceshowed statistically significant reduction (>50%) in LDL cholesterolvalues. This data is provided in FIG. 27.

Example 13

This example demonstrates atherosclerotic changes that ApoE−/− miceexperienced when treated with KGYY6 (6-mer—SEQ ID NO: 4). ApoE−/− micewere fed a high fat diet for 16 weeks. Mice were randomly assigned toreceive dose of 1 mg/kg of KGYY₆ (6-mer—SEQ ID NO:4) by IV tailinjection, once a week for a period of 4 weeks. Control mice receivedvehicle only. Atherosclerosis was investigated by several methods.En-face analysis utilizing Sudan IV stain (lipid stain) demonstrated asignificant reduction in lesion areas. FIG. 28A is an image of KGYY6treated aortic en-face Sudan IV staining and FIG. 28B is an image ofcontrol (untreated) aortic en-face Sudan staining. FIG. 29 is a graphdemonstrating the reduction of lesion areas of Sudan IV staining.

Further, measurement of plaque area and morphology was performed usingthe Paigen method. This method obtains sequential 5 um aortic crosssections from the aortic root beginning at the valve leaflets into theascending aorta. At 50 um intervals, slides are stained after which thearea of atherosclerotic lesion is measured. Individual plaque areameasurements are plotted against the micrometer intervals and a curve isestablished, with the area under the curve (AUC) giving the total volumeof plaque. These results are presented in graph format in FIG. 30, whichdemonstrates that mice treated with KGYY6 showed reduction in plaquevolume under the curve. Moreover, characterization of plaque compositionor aortic samples was performed using trichrome staining techniques andthese results are presented in graph format in FIG. 31. Indeed, thisFIG. 31 data, which was generated using Image Pro Plus softwareanalysis, quantifies and shows that plaque compositional changesoccurred, including increased collagen and reduced smooth musclecontent.

FIG. 32A is an image of trichrome stained cells of the cross-sections ofthe aorta of the KGYY6 treated subject. FIG. 32B is an image oftrichrome stained cells of the cross-sections of the control subjects.In FIG. 32A, 50 identifies areas characteristic of plaque formation. 60identifies aortic valve leaflets. FIG. 32B, the control (untreated) 50areas characteristic of plaque (area under the curve) are greater thanthose in the subjects treated with the KGYY6 peptide.

Example 14

This example demonstrates that Th40 cell counts in T1D patients withhigh calcium scores may be higher than the Th40 cell counts in T1Dpatients with low calcium scores. Accordingly, using this data may allowa practitioner to determine whether a T1D patient might benefit fromtreatment with the peptide based on Th40 cell levels. Moreover, thisdata may allow a practitioner to confirm or rule out subjects withatherosclerosis.

Peripheral blood was obtained from human patients for three differentsample populations. These populations included: (1) patients with T1Dwith low Coronary Artery Calcium (CAC) scores, (2) patients with T1Dwith high CAC scores, and control populations. The peripheral blood wasmeasured for total count of CD40+CD4+ cell numbers as shown in FIGS. 33Aand 33B. Flow cytometry was performed utilizing a MACSQuant® Analyzer 10(Miltenyi Biotec Inc.). Additional analysis was performed using FlowJo ®(FlowJo, LLC wholly owned by BectonDickinson, Inc.) single-cell flowcytometry software. VioBlue A was used as a CD40 stain (Y-axis in bothFIG. 33A and 33B). APC Cy7 used as CD4 stain (X-axis in both FIG. 33Aand 33B).

FIG. 33A shows the FlowJo® analysis for T1D patients with high calciumscores. Quadrant 2 (Q2) represents the Th40 cell population (CD4+CD40+).Quadrant 3 (Q3) represents the population of CD40-CD4+. FIG. 33B showsthe FlowJo® analysis for T1D patients with low calcium scores. A higherpercentage of Th40 cell population (CD4+CD40+) may be observed in T1Dpatients with high calcium scores as in FIG. 33A compared to a lowerpercentage of Th40 cells (CD4+CD40+) in T1D patients with low calciumscores as in FIG. 33B.

FIG. 34A is a chart of calcium scores versus percent Th40 cells indifferent patient populations. The percent of Th40 cells issignificantly increased in patients with T1D with high Coronary ArteryCalcium (CAC) scores compared to both T1D patients with low CAC scoresand control populations. FIG. 34B is an alternative chart demonstratingthe individual variability of the patients that made up each of thethree different populations tested.

Example 15

This example demonstrates that Th40 cell counts in T1D patients withhigh calcium scores may be higher than the Th40 cell counts in T1Dpatients with low calcium scores. This data may allow a practitioner toconfirm or rule out subjects with atherosclerosis.

Peripheral blood was obtained from human patients for three differentsample populations. These populations included: (1) patients with T1Dwith low Coronary Artery Calcium (CAC) scores, (2) patients with T1Dwith high CAC scores, and control populations. The peripheral blood wasmeasured for total count of CD40+CD4+ cell numbers as shown in FIG. 35and FIG. 36. In this study, human subjects age and sex matched, weresamples from the Coronary Artery Calcification in Type 1 Diabetes Study.After flow cytometry of lymphocytes, high and low calcium scores wereunblinded. The high calcium score patients have consistently higher Th40cell percentage, with a statistically significant increase in interferongamma (IFNy) compared to low calcium score patients.

FIG. 35 is a is a graph of percentage of Th40 cells in low coronaryartery calcium (CAC) and high coronary artery calcium (CAC) subjects.FIG. 36 is a graph of the percentage of interferon gamma (IFNy) in Th40cells in low coronary artery calcium (CAC) and high coronary arterycalcium (CAC) subjects.

Example 16

This example demonstrates administration of the KGYY₆ (6-mer—SEQ IDNO:29) affects the inflammatory cytokine production of IL2, INFγ, andIL17a.

Spleens from ApoE mice and C57BL/6 mice were processed for lymphocytes.ApoE−/− and C57BL/6 mice were fed a 60% high fat diet (research diet)for 1 week. Splenic lymphocytes were treated with 6-mer in vitro for 24hours. Cells were placed in media overnight in the presence of varyingconcentrations of 6-mer peptides. The following morning, Brefeldin A wasadministered for 4 hours. All cells were stained for CD3, CD4, CD40(Th40 cells) and measured by use of flow cytometry for their productionof IL2, INFγ, and IL17a. The results of this study are shown in FIG. 37.

Example 17

Controlling the Residual Inflammatory Risk in Type 1 DiabeticAtherosclerosis

Prevention trials with statins reduce the relative risk ofcardiovascular (CV) events by 10 to 40%. This leaves a ‘residual risk’of 60-90% for which the CANTOS trial (ILlb inhibition) provided proof ofconcept that targeting inflammation reduces CV event rates;unfortunately, it did not change rates of type 1 diabetes (T1D) andinfections in T1D patients were higher. A major molecular driver ofauto-inflammation in both diseases is the CD40/154 inflammatory dyadwith unique T cell subset, CD3+CD4+CD40+ (TH40 cell) known to besignificantly expanded in T1D subjects. Normalization of the aberrantcontact dependent interaction of this dyad has shown efficacy in animalmodels using antibody to CD154, reversing T1D and rate ofatherosclerosis, but unfortunately has deleterious side effects whengiven in humans.

This Example 17 measured TH40 cells in T1D subjects with high calciumscores (a measure of atherosclerotic burden) and those with low calciumscores. Additional to the higher percentage of TH40 cells compared tocontrols, this Example 17 found a statistically significant elevation inin INFγ production in the high calcium score cohort, indicating a morepathogenic state of these cells.

Peptides were designed that are capable of binding directly to the CD40receptor which have been shown to reverse T1D in animals. These peptideswere administered to ApoE−/−mice, a model of atherosclerosis.Immunohistochemical staining demonstrated significant reduction inplaque as well as smooth muscle and collagen content. In-vitro analysisof TH40 mouse splenic cells demonstrated modulation in IL2, IFNγ andIL17, all potent cytokines of atherosclerosis.

This new information relays promise for a more directed diagnostic andtherapeutic target for T1D related atherosclerosis.

Background

In primary and secondary prevention trials, statins reduced the relativerisk of major CV events by 10 to 40%, leaving a ‘residual risk’ of60-90% of events (Fruchart, J. C., et al. Cardiovasc. Diabetol. 2014;13:26; Fruchart, J. C., et al., The American Journal of Cardiology,2008; 102(10 Suppl):1K-34K). Of this residual risk, those with diabetesstill carry substantial residual ‘inflammatory’ risk despite optimalmedical therapy (Wong, N. D. et al., J Clin. Lipidol., 2017,11(5):1223-33. Epub2017/06/30; Ridker, P. M., European Heart Journal,2016; 37(22):1720-2; Ridker, P. M., Circulation Research, 2017,120(4):617-9). Clinical and experimental data support an additionalcritical role for the treatment of inflammation in atherosclerosis(Sigala, F., et al. Curr. Opin. Pharmacol., 2017; 39:9-18.Epub2017/12/01; Antonopoulos, A. S. et al., Curr. Opin. Pharmacol.,2017;39:1-8). Multiple studies in human diabetes, including autoimmuneT1D and T2D, have demonstrated a relative increase in soluble CD154(Jinchuan, Y., et al., Clin. Chim. Acta., 2004; 339(1-2):85-90).

CD40 expressing T cells are not only necessary and sufficient totransfer Type 1 Diabetes in animal models (Waid, D. M. et al., Eur. J.Immunol., 2004; 34(5):1488-97; Wagner, D. H., et al., Proc. Natl. Acad.Sci. USA, 2002; 99(6):3782-7) but translational studies showed thatthese cells are significantly increased in number in human subjects,reflective of auto-inflammation, compared to non-autoimmune controls(Waid, D. M., et al., Clin. Immunol., 2007; 124(2):138-48). Numerousstudies show that blocking CD40 prevents inflammation and prevents orimproves a variety of autoimmune diseases, including atherosclerosis andrecently T2D (Poggi, M. et al., Diabetologia. 2009; 52(6):1152-63; PoggiMetal., Arterioscler. Thromb. Vasc. Biol., 2011; 31(10):2251-60;Desai-Mehta A., et al., J Clin. Invest., 1996; 97:2063-73). Whether dueto the increase in Th40 or increase in CD154 in those with T1D or T2D,modification of its interaction has proven to be a prime target forcontrolling residual inflammatory risk.

Methods

Human subjects were sampled from the CACTI study (Coronary ArteryCalcification in Type 1 Diabetes Study). In blinded fashion, fresh bloodfrom T1D patients with high and low calcium scores were obtained. Bloodwas processed for lymphocytes and analyzed using flow cytometry for TH40cells and cytokines.

Design of the KGYY15 peptide. (A) Based on the amino acid sequence ofthe murine CD154 sequence (the human sequence is >90% homologous) aseries of peptides was generated. The relative peptide stabilityassessed by ExPaSYanalysis is shown in the table below.

Stability as Assessed by Length Sequence ExPaSy Analysis 24-merAASVLQWAKKGYYTMKSNLVVLEN    2 min. 15-mer VLQWAKKGYYTMKSN >100 hrs.13-mer VLQWAKKGYYTMK   30 hrs. 10-mer WAKKGYYTMK    2 hrs. 8-merAKKGYYTM    1 hr. 6-mer AKKGYY   30 min.

KGYY15 and KGYY6 prevents diabetes onset in NOD mice. NOD mice at 6weeks of age were injected weekly iv with 25 ug (1 mg/kg) of eachpeptide through 45 weeks. Each cohort included 10 animals. Bloodglucoses were measured and levels at 250 mg/dl or higher for 3consecutive measures were considered diabetic. The RGD peptide is a 15amino acid sequence from the CD154 sequence that does not include theCD40 binding motif. (This is showing in FIG. 1).

ApoE−/−mice (n=16, 4 male and 4 female per group), fed a 60% high fatdiet (Research Diets) for 16 weeks. 8 mice were injected with 1 mg/kgKGYY6 and 7 with vehicle alone. Glucose tolerance testing (GTT) wasperformed by fasting 6 hours, followed by intraperitoneal injection of 1g/kg body weight glucose in water. Both blood glucose and serum insulinwere measured at 0, 15 min, 30 min, 60 min and 2 hours.

Spleens from ApoE mice and C57BL/6 mice were processed for lymphocytes,placed in media overnight in the presence of varying concentrations ofKGYY6 peptide. BrefeldinA was administered for four hours. All cellswere stained for CD3, CD4, CD40 (Th40 cells) and measured by flowcytometry for production of IL2, IFNg, and IL17a (each demonstratingvarying degrees of reduction).

Results

Human Data. Human subjects, age and sex matched, sampled from CoronaryArtery Calcification in Type 1 Diabetes (CACTI) Study. After flowcytometry of lymphocytes, high and low calcium scores were unblinded.The high calcium score patients appear to have consistently higher TH40cell percentage, with a statitistically significant increase ininterferon gamma compared to low calcium score patients.

Peptide Data. CD3+CD4+CD40+ splenic cells treated with varyingconcentrations of peptide, each demonstrating varying degrees ofreduction in IL2, INF gamma and IL17a.

Mouse Data. There were no changes in starting values of weights, glucoseor lipid values (data not shown). FIGS. 28-32B provide additionalshowings of atherosclerotic changes of mice treated with KGYY 6 andcontrol mice.

Conclusions

We have noted CD3+CD4+CD40+(TH40) cells are increased in T1D patients.They are known to be both necessary and sufficient to cause T1D in mice.CD40 is also known to be atherogenic. Not all type 1 diabetic patientshave significant atherosclerosis though they carry a high risk of suchan occurrence.

TH40 cells appear to be consistently higher in diabetic patients withhigh calcium scores compared to those with low calcium scores and appearto be more actively producing INFγ.

With residual inflammatory risk having emerged as a therapeutic targetand, given the importance of controlling CD40 as a therapeutic approachto inflammation, we created a novel approach to modulate CD40 and arecurrently utilizing this method in the control of autoimmuneinflammation.

This peptide was designed on the CD154 protein sequence that encompassknown hot spots for receptor-ligand interaction and has demonstrated:

Reduction in IL2, IFNg, and IL17a in treated mice.

Reduction in atherosclerotic lesion area and volume.

Plaque compositional change.

From the foregoing, it is readily apparent that new and usefulimplementations of the methods have been herein described andillustrated which fulfill numerous desiderata in remarkably unexpectedfashions. It is, of course, understood that such modifications,alterations and adaptations as may readily occur to the artisanconfronted with this disclosure are intended within the spirit of thisdisclosure, which is limited only by the scope of the claims appendedhereto.

What is claimed:
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 50. A method to confirm or rule out diagnosis of cardiovascular disease, atherosclerosis, and/or coronary artery disease in a patient comprising: determining the percentage of Th40 cells in a sample isolated from a subject presenting with Type 1 Diabetes, comparing the percentage of Th40 cells to a control sample or a standard value, and, diagnosing cardiovascular disease and/or coronary artery disease in the subject having an increase in the percentage of Th40 cells in the sample from the subject relative to the control sample or standard value; or, ruling out cardiovascular disease, atherosclerosis, and/or coronary artery disease in the subject having no increase or a decrease in the percentage of Th40 cells in the sample from the subject relative to the control sample or standard value.
 51. The method of claim 50, wherein the percentage of Th40 cells in the sample is assessed by staining with a labeled antibody that specifically recognizes a protein selected from CD40, CD4, CD8, CD25, and/or CD45 and analyzing the stained cells by flow cytometry to determine the percentage of stained cells in the sample.
 52. The method of claim 50, wherein the percentage of Th40 cells in the sample is assessed by staining with a labeled antibody that specifically recognizes a protein selected from CD40 and CD4 and analyzing the stained cells by flow cytometry to determine the percentage of stained cells in the sample.
 53. The method of claim 50, wherein the control sample is a sample from at least one subject known not to have cardiovascular disease, atherosclerosis, and/or coronary artery disease.
 54. The method of claim 50, wherein the control sample is a sample from at least one subject known to have cardiovascular disease, atherosclerosis, and/or coronary artery disease.
 55. The method of claim 50, wherein the control sample is a sample from at least one subject known to have type 1 diabetes and a low coronary artery calcium (CAC) score.
 56. The method of claim 50, wherein the control sample is a sample from at least one subject known to have type 1 diabetes and a high coronary artery calcium (CAC) score.
 57. The method of claim 50, wherein the control sample is a baseline sample obtained from the subject at an earlier date.
 58. The method of claim 50, wherein the sample is whole blood, plasma, serum, or a subfraction of whole blood.
 59. The method of claim 50, wherein the method further comprises determining the percentage of cells expressing additional markers selected from CD4, CD40, CD8, CD25, CD45, TCRV8.3+, CDCR3, and CCR5.
 60. A method to confirm or rule out diagnosis of cardiovascular disease, atherosclerosis, and/or coronary artery disease in a patient comprising: determining the percentage of Th40 cells in a sample isolated from a subject presenting with symptoms suggestive of cardiovascular disease, atherosclerosis, and/or coronary artery disease, comparing the percentage of Th40 cells to a control sample or a standard value, and diagnosing cardiovascular disease, atherosclerosis, and/or coronary artery disease in the subject having an increase in the percentage of Th40 cells in the sample from the subject relative to the control sample or standard value is indicative of cardiovascular disease, atherosclerosis, and/or coronary artery disease in the subject.
 61. The method of claim 60, wherein the percentage of Th40 cells in the sample is assessed by staining with a labeled antibody that specifically recognizes a protein selected from CD40, CD4, CD8, CD25, and/or CD45 and analyzing the stained cells by flow cytometry to determine the percentage of stained cells in the sample.
 62. The method of claim 60, wherein the percentage of Th40 cells in the sample is assessed by staining with a labeled antibody that specifically recognizes a protein selected from CD40 and CD4 and analyzing the stained cells by flow cytometry to determine the percentage of stained cells in the sample.
 63. The method of claim 60, wherein the control sample is a sample from at least one subject known not to have cardiovascular disease, atherosclerosis, and/or coronary artery disease.
 64. The method of claim 60, wherein the control sample is a sample from at least one subject known to have cardiovascular disease, atherosclerosis, and/or coronary artery disease.
 65. The method of claim 60, wherein the control sample is a sample from at least one subject known to have type 1 diabetes and a low coronary artery calcium (CAC) score.
 66. The method of claim 60, wherein the control sample is a sample from at least one subject known to have type 1 diabetes and a high coronary artery calcium (CAC) score.
 67. The method of claim 60, wherein the control sample is a baseline sample obtained from the subject at an earlier date.
 68. The method of claim 60, wherein the sample is whole blood, plasma, serum, or a subfraction of whole blood.
 69. The method of claim 60, wherein a peptide selected from the group of SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9 and/or SEQ ID NO:10, is used to determine the percentage of Th40 cells in a sample isolated from a subject.
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